FACS Laboratory
[email protected]
Training Guide
http://london-research-institute.org.uk/technologies/facs
BD LSRs
Operator Training
LRI FACS lab
1 Components
1.1 Basic parts overview……………………………………………………………………………………….pg3-6
Overview of LSRs…………………………………………………….……………………3
Filling and emptying the tanks…………………………………..…………..……5
Starting up the LSR…………………………………………………………….………..6
Cleaning and Shutting down…………………………………………………………6
1.2 Fluidics…………………………………………………………………………………………………………….pg7-11
Basic fluidic mechanics in flow cytometry ……………………………………7
The sample injection port (SIP) …………………………………………………..9
Troubleshooting…………………………………………………………………………….10
o
Unblocking a probe……………………………………………………………10
o
De-bubbling filters……………………………….……………………………11
1.3 Optics……………………………………………………………………………………………………………...pg12-31
Optics Overview.……………………………………………………………………………12
Detection optics.……………………………………………………………………………13
1.4 Digital electronics………………………………………………………………………………………………pg32-34
Digital flow cytometry and the LSRs…………………………………………….32
Digital pulse processing..………………………………………………………………32
2 Fluorochromes & Multi-colour Flow
2.1 Commonly used fluorochromes………………………………………………………………………..pg35-39
2.2 Fluorochrome selection……………………………………………………………………………………..39-41
Viability dyes……………………………………………….………………………………….39
Fixable LIVE/DEAD amine reactive dyes……………………………………….40
Availability………………………………………………………………………………………40
Physical Characteristics………………………………………………………………….41
2.3 Combining fluorochromes………………………………………………………………………………….pg41-43
Spectral overlap………………………………………………………………………………41
Problem pairs.…………………………………………………………………………………41
Example combinations……………………………………………………………………42
2.4 Compensation…………………………………………………………………………………………………….pg44-49
Controls.…………………………………………………………………………………………44
Compensation beads.……………………………………………………………………45
The bi-exponential display.……………………………………………………………47
Compensation - the median fluorescence technique..…………………37
Copying spectral overlap.………………………………………………………………49
Automatic compensation………………………………………………………………..49
3 BD FACSDivaTM : Software Basics
3.1 The Browser, Worksheet, Inspector and Acquisition windows………………………..pg50-52
3.2 Example experiment………………………………………………………………………………………….pg52-73
Working with plots, regions and gates.…………………………………………55
Baseline PMT voltages……………………………………………………………………62
Compensation…………………………………………………………………………………63
o
Automatic compensation……………………………………………..…….68
Exporting data from FACSDiva as listmode (.fcs) files…………………72
Working with templates…………………………………………………………………73
4 Quality Control
4.1 Routine Checks..…………………………………………………………………………………………………pg74-75
4.2 Laser Delay, Area Scaling and Windows Extension………………………………………….pg75-76
5 Sample preparation
5.1 Harvesting cells………………………………………………………………………………………………….pg77
5.2 Resuspending cells for analysis…………………………………………………………………………pg78
6 Glossary.………………………………………………………………………………………………………………pg79-84
7 Troubleshooting Guide .……………………………………………………………………………………pg85
1.1 Basic Parts Overview
In the FACS Lab, all cytometers are normally switched on at 9am. When booking to start
before or at 9, or on weekends, please be aware that the LSRs have multiple laser lines
that need to be given a minimum of 30 minutes to warm up before running samples.
Overview of LSRs
From the control panel you can perform the operations RUN, STANDBY and PRIME, and
make some sample flow rate adjustments. The machine should be on STANDBY with a
tube of water when you come to it. Check that the sheath tank is filled to the line and
the waste tank is empty. If the sheath tank runs dry, air will enter the system and can
be difficult to remove; you will be unable to acquire any data if this happens.
* Important! *
Before starting an experiment, check the levels of sheath fluid and waste. If you are
using the machine for a long time check the tanks regularly. We cannot stress enough
how critical it is that you are capable of maintaining the tanks and purging air from the
system. If you are unsure of how to do so, please ask a member of the FACS Lab, to
avoid ruining an important experiment, and possibly the next user’s experiment too!
LSRIIA and LSRIIB overview
1. Power switch
2. Sheath tank
3. Waste tank
4. Control panel
5. Sample Injection
Port (SIP)
6. Sheath Filter
7. Sheath line air
purge (roll-valve)
Fig. 1.1.1 Location of important parts on LSRII.
3
LSRFortessa A, B and C overview
1. Power switch
2. Sheath tank
3. Waste tank
4. Control panel
5. Sample Injection
Port (SIP)
6. Sheath filter
7. Sheath line air
purge (roll-valve)
Fig. 1.1.2 Location of important parts on LSRFortessa
4
Filling and emptying the tanks
A
B
Fig. 1.1.3 A De-pressurising sheath tank by pulling up on valve (black circle). B Lid of
waste tank showing waste line (orange tube) and alarm (black wire).
First check the machine is in STANDBY (standby button on control panel is orange):
*Filling the sheath tank* Fig. 1.1.3 A de-pressurising tank
Unclip the sheath pressure line (red circle - green tube on top of tank).
De-pressurise the tank (black circle - pull metal release valve up).
Unscrew & remove lid.
Fill with PBS to groove in tank - do not overfill.
PBS can be located in bottles on the shelf in the corridor.
Replace the lid and re-connect the pressure line.
De-bubble the LSR – it is good practice to always do so when you re-fill the tank.
Please refer to the Troubleshooting section in section 1.2 for instructions.
*Emptying the waste* Fig. 1.1.3 B waste tank lid
Unclip the orange line (waste line) and disconnect the alarm (black wire - twist
connection) from the lid of the waste tank.
Unscrew the lid: add 100g Virkon powder (use measuring cup in Virkon container) to
the tank. Empty the waste into the sink by LSRIIA (Virkon is kept under the sink).
Replace lid, re-connect waste line and alarm.
5
Starting up the LSR
Switch on the LSR and start the computer workstation. There is no password to log into
Windows (hit “Enter”).
Priming the machine
Before running any samples it is good practice to prime the fluidics to remove any
bubbles from the flow cell. Additionally, before you run your sample (or while you are
waiting for the lasers to warm up), it is a good idea to check the machine is clean by
simply running fresh dH20 in a new tube. If move arm to side during Prime, remember to
return it after! When running dH20, have threshold at 15,000 and FSC at voltage used
for cells. The event rate should be mostly 0 events/second but up to 100evt over 30
seconds due to bubbles, noise. If you have time, it does not hurt to run some cleaning
agents for a short period, followed by dH20. After these steps and allowing the lasers to
warm up, the machine is ready to run your samples - this will be covered later.
*Priming the LSR*
Remove the tube of dH20 from the sample injection port (SIP).
With SIP arm to the side, press PRIME; this empties out the flow cell and refills it to
remove bubbles. LSR returns to STANDBY when done. Repeat 1-2 times.
Cleaning and shutting down
When you are done running your samples, it is imperative to clean the LSR sufficiently to
prevent clogging of the SIP and remove dyes which may remain in the tubing:
*Cleaning procedure*
Run tube of detergent for 5 min on HI (BD FACS Rinse).
Run tube of bleach (BD FACS Clean) for 3min on HI if DNA dyes/viability dye such as
DAPI, Hoechst, PI etc. have been used; if not - proceed to next step.
Run tube of dH20 for 5min.
Check the machine is clean with a fresh tube of dH20.
If machine is not clean, repeat Clean & Rinse steps.
If the machine has been left dirty, running cleaning agents for a longer length of time
will usually help; otherwise the machine may require a long clean which is routinely
performed by the FACS Lab staff.
The last user of the day should switch off the cytometer after the cleaning.
IMPORTANT! Read also Troubleshooting, in Section 1.2 - purging air from the machine.
6
1.2 Fluidics
A pressurised fluidics system delivers the sample to the flow cell where interrogation by
the lasers takes place, using the process of hydrodynamic focusing to help create a
tight, single cell flow to allow us to look at each cell one by one. This is achieved by
laminar flow, higher sheath to core velocities and by the shape of the flow cell.
Flow cell
Lasers
Sample
Lo/Med/Hi
controls
Vacuum
Waste
Sheath
Pressure line
Fig. 1.2.1 Basic diagram of flow cytometer fluidics system
Basic Fluid Mechanics in Flow Cytometry
In the cuvette type cytometers (e.g. Caliburs, LSRs), cells are introduced into the middle
of the flow cell, injected into the centre of a smoothly flowing stream (the sheath fluid).
Since both the core stream (consisting of your injected sample) and the sheath stream
are flowing smoothly, laminar flow is observed where the two will maintain their relative
positions and do not mix.
We want to analyse one cell at a time, so a narrow core carrying single cells to the laser
interrogation point(s) is critical. One of the ways this is achieved is by using a cuvette
where the sides taper inwards towards the area of laser interrogation.
7
Note that fluid flow at the exit from the
region of cuvette constriction into the
narrower region where laser interrogation
occurs can be described as slug flow
(having constant velocity across the fluid
cross section). This continues for a while,
but quickly returns to a parabolic laminar
flow profile (velocity at the edges is
slower than at the centre).
Fig. 1.2.2 Fluid dynamics in the flow cell.
(Shapiro’s Practical Flow Cytometry, 4th Ed.)
It is important that laser interrogation
occurs before the parabolic profile is reestablished so that cells will have the
same velocity - and therefore take the
same time to pass through the laser
beam, regardless of the position within
the core. In other words, variations in
distribution will not have an impact on
illumination
time,
giving
better
instrument precision.
(Further detail can be found in Howard Shapiro’s Practical Flow Cytometry 4th Ed).
Which speed to run your sample- HI, MED, LO?
Although we cannot adjust the pressure of the sheath, we can change the pressure
applied to the sample, to inject more/less sample per unit of time. This is useful when
the sample is very dilute or to save time when looking for very rare events (i.e. need to
run through a large number of cells).
Increasing the sample pressure will result in a widening of the core carrying your
particles of interest, as shown in Fig. 1.2.3. This affects how uniformly particles flow past
the interrogation points and will affect measurements such as DNA (which are best run
in LO) where precision is important. Increasing sample pressure will result in an increase
in DNA histogram CV.
For qualitative measurements such as immuno-phenotyping, there may be some loss of
resolution but running in MED/HI is generally not a problem, when using log scale.
8
Fig. 1.2.3 Core stream diameter increase is observed when sample pressure is high
(right) compared to low sample pressure (left).
Samples should be sufficiently concentrated to enable a reasonable run time, especially
if you need to run your samples in LO. Correct sample preparation (see section 5) is also
vital to prevent blocking the probe: there is no PAUSE function in DIVA, one must stop
recording, clear the blockage, then APPEND to the saved file. It is better to avoid having
clumps in the first place.
The sample injection port (SIP)
The SIP comprises the sample injection tube and support arm. The sample injection tube
is encased in a sleeve. When the support arm is open, the sample injection tube backflushes sheath fluid. This is then removed by a vacuum pump (activated when the arm is
open) up the outer sleeve to the waste tank, helping to clean the injection tube between
samples.
Fig. 1.2.4 Sample injection port component diagram and photograph.
9
*Important!*
The machine starts taking up your sample as soon as you have installed the tube on
the SIP, NOT when you press Acquire.
Also, leaving a sample on the injection tube with the support arm open will
result in loss of sample! Be quick with the support arm when putting tubes in place.
Troubleshooting
Unblocking a probe
Properly prepared samples should not block the
machine. Samples should be carefully checked for
clumps that can block the probe (filter and/or pipette
sample if ANY clumps visible).
Check first that the sheath tank is not empty:
Remove your sample and Prime several times. If
this does not work, run Clean followed by Rinse for
5-10 minutes. Run dH20 for 2-3 minutes before
putting your sample back on.
Fig. 1.2.5 Unblocking the
sample probe using a syringe.
For a persistent clog, it is sometimes necessary to apply a gentle vacuum by syringe there is a large syringe with rubber tubing in the lab for this purpose. Please ask a
member of staff to demonstrate.
Make sure that when you prime the machine, there is no tube in place (first, the flow cell
empties- flushing out the probe, then the flow cell re-fills). Keeping a tube of
Rinse/Clean/dH20 on as you do this is counter-productive, as any debris flushed out will
also be taken back up again.
*Problematic samples with clumps*
Please be aware that if you can see the clump, it is too big!
10
De-bubbling filters
If the machine has been allowed to run dry you may need to purge air from the filter on
the sheath tank and sheath line, and also the flow cell (using Prime). Air can be hard
to remove; it is better to prevent this from happening in the first place by checking the
tanks!
*Knowing how to troubleshoot is important to avoid running into
trouble if you will be using the machine after hours*
You may be left unable to run the rest of your experiment, or cause subsequent
users to be unable to run theirs.
This problem may present itself as unexpected dots on your plots (caused by air
bubbles, includes both randomly distributed events and low scatter “debris-like” signals)
accompanied by the LSR not showing events from your tube.
De-bubbling Filters
A
B
Fig. 1.2.6 A Purging air from sheath filter: Tap filter gently and check for bubbles.
Carefully roll valve and PBS is released along with bubbles. Tilting the tank/filter as you
do this in order to encourage the bubbles to move towards the purge valve can also be
useful, please ask a member of staff to demonstrate. B Purging air from the sheath
line: roll valve (PBS is released, along with bubbles). The cytometer should be on
standby while de-bubbling filter/line.
11
1.3 Optics
Optics Overview
Excitation:
Light sources: lasers
LSRIIA - Blue (488nm), Violet (405nm), UV (355nm), Red (633nm)
LSRIIB - Blue (488nm), Violet (405nm), Yellow-Green (561nm), Red (633nm)
Fortessa A - Blue (488nm), Violet (405nm), Yellow-Green (561nm), Red
(633nm), UV (355nm)
Fortessa B - Blue (488nm), Violet (405nm), Yellow-Green (561nm), Red
(639nm), UV (355nm)
Fortessa C - Blue (488nm), Violet (405nm), Yellow-Green (561nm), Red
(639nm), UV (355nm
Filters/mirrors: Route the laser beam to the flow cell to interrogate the sample.
Collection: Scattered light and any emitted fluorescence are directed by fibre optics to
the appropriate block of photomultiplier tubes (PMTs or ‘detectors’) specific for each
laser.
Filters/mirrors: Used to guide the light to the appropriate PMT in the block.
Detectors: Receive the scattered light and fluorescence signals. From these photons,
an amplified electrical signal is generated that we observe through the Diva
software.
Fig. 1.3.1 Example diagram of LSRII optical bench
12
The optical bench of the LSRs see a departure from the layout seen in the FACS Scan,
Calibur and Vantage. Instead of passing the light through several mirrors to the correct
PMT, the fluorescence from each laser (spatially separated) is directed to the relevant
octagonal or triagonal arrangement of PMTs. This results in more efficient collection of
the signals.
Detection Optics
The filters used in the cytometers are optimized for their respective detectors. They can
be changed but their transmission characteristics should be known. Gloves should be
worn when handling the filters and dichroic mirrors, and they should be held by their
edges to avoid leaving fingerprints on the transmission surfaces. The specifications of
the filters can be found on the outside edge (by which they should be handled) in small
print. A short pass filter (SP), for example 450SP, transmits wavelengths shorter than
its specification- i.e. 450nm. Long pass (LP) filters are similarly named, for example
610LP, which transmits light longer than 610nm. Band pass (BP) filters transmit within
a given range and are named accordingly. For example, a “710/50BP” is a filter that
transmits 710nm±25nm (685nm-735nm), “530/30BP” transmits 515-545nm.
Dichroic mirrors can also have a short or long pass function, transmitting longer/shorter
than the specification. These are also known as beam-splitters as the rest of the light is
reflected. Dichroics are particularly useful for directing light of specific wavelengths down
the appropriate route within the optical bench.
In front of each PMT is a dichroic long pass (DCLP) mirror. At the first PMT, the longest
wavelength to be analysed passes through and everything shorter is reflected to the
next PMT. At the second PMT, there is a similar arrangement with DCLP allowing the
next longest wavelength to pass and reflecting the rest; and so forth down the PMTs.
This set up loses less light than the older collection optics (about 10% is lost passing
through the dichroic- generally, mirrors are more efficient at reflecting than filters are at
transmitting light).
13
Fig.1.3.2 Filter processing of light to allow specified wavelengths to be transmitted.
The standard configuration of filters is listed inside the hood of the LSRII; it is a good
idea to check that the person before you has not changed the filters without returning
them to the standard set-up. Please ask a member of the FACS Lab to demonstrate how
to change optical filters before attempting to do so yourself.
LSRIIA PMT Position
LSRIIA Diva Parameter
Blue octagon, A
780/60 blue
Longpass Filter
735DCLP
Blue octagon, B
695/40 blue
680DCLP
Blue octagon, C
660/20 blue
635DCLP
Blue octagon, D
610/20 blue
595DCLP
Blue octagon, E
575/26 blue
550DCLP
Blue octagon, F
530/30 blue
505DCLP
Blue octagon, G
SSC
-
FSC
FSC
-
Violet trigon, A
530/30 violet
505DCLP
Violet trigon, B
450/50 violet
UV trigon, A
525/50 UV
450DCLP
UV trigon, B
440/40 UV
-
Red trigon, A
Red trigon, B
780/60 red
730/45 red
755DCLP
Red trigon, C
660/20 red
-
DCLP = Dichroic Long Pass mirror.
Fig. 1.3.3 Standard optical filter configuration in LSRIIA.
14
685DCLP
Blue octagon, A
LSRIIB Diva
Parameter
695/40 blue
Blue octagon, B
575/26 blue
550DCLP
Blue octagon, C
530/30 blue
505DCLP
Blue octagon, D
SSC
-
Blue octagon, E
-
-
Blue octagon, F
-
-
Blue octagon, G
-
-
FSC
FSC
-
Violet trigon, A
660/20 violet
635DCLP
Violet trigon, B
605/40 violet
595DCLP
Violet trigon, C
560/40 violet
550DCLP
Violet trigon, D
510/20 violet
505DCLP
Violet trigon, E
450/50 violet
-
Violet trigon, F
-
-
Y/Green octagon, A
780/60 yellow
735DCLP
Y/Green octagon, B
705/70 yellow
690DCLP
Y/Green octagon, C
660/20 yellow
635DCLP
Y/Green octagon, D
620/40 yellow
600DCLP
Y/Green octagon, E
585/15 yellow
570DCLP
Y/Green octagon, F
-
-
Red trigon, A
780/60 red
735DCLP
Red trigon, B
710/50 red
685DCLP
Red trigon, C
660/20 red
-
LSRIIB PMT Position
DCLP = Dichroic Long Pass mirror.
Fig. 1.3.4 Standard optical filter configuration in LSRIIB.
15
Longpass Filter
685DCLP
Fortessa A PMT Position
Blue octagon, A
Fortessa Diva Parameter
780/60 blue
Longpass Filter
750DCLP
Blue octagon, B
695/40 blue
685DCLP
Blue octagon, C
610/20 blue
600DCLP
Blue octagon, D
580/30 blue
550DCLP
Blue octagon, E
530/30 blue
505DCLP
Blue octagon, F
SSC
-
FSC
FSC
-
Violet octagon, A
530/30 violet
475DCLP
Violet octagon, B
450/50 violet
-
UV trigon, A
530/30 UV
505DCLP
UV trigon, B
450/50 UV
-
Red trigon, A
780/60 red
750DCLP
Red trigon, B
730/45 red
710DCLP
Red trigon, C
670/14 red
-
Y/Green octagon, A
780/60 yellow
750DCLP
Y/Green octagon, B
710/50 yellow
685DCLP
Y/Green octagon, C
670/30 yellow
635LP
Y/Green octagon, D
610/20 yellow
600DCLP
Y/Green octagon, E
582/15 yellow
-
DCLP = Dichroic Long Pass mirror.
Fig. 1.3.5 Standard optical filter configuration in LSRFortessa A.
16
Fortessa B and C PMT
Fortessa Diva Parameter
Longpass Filter
Blue octagon, A
710/50 blue
685DCLP
Blue octagon, B
530/30 blue
505DCLP
Blue octagon, F
SSC
-
FSC
FSC
-
Violet octagon, A
670/30 violet
630DCLP
Violet octagon, B
610/20 violet
600DCLP
Violet octagon, C
586/15 violet
570DCLP
Violet octagon, D
540/30 violet
535DCLP
Violet octagon, E
525/50 violet
505DCLP
Violet octagon, F
450/50 violet
-
UV trigon, A
450/50 UV
505DCLP
UV trigon, B
530/30 UV
-
Red trigon, A
780/60 red
750DCLP
Red trigon, B
730/45 red
690DCLP
Red trigon, C
670/14 red
-
Yellow/Green octagon, A
780/60 yellow
750DCLP
Yellow/Green octagon, B
710/50 yellow
685DCLP
Yellow/Green octagon, C
670/30 yellow
635LP
Yellow/Green octagon, D
610/20 yellow
600DCLP
Yellow/Green octagon, E
586/15 yellow
-
Position
DCLP = Dichroic Long Pass mirror.
Fig. 1.3.6 Standard optical filter configuration in LSRFortessa B and C.
17
LSRII (A) Blue Laser Octagon: filter optics and collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.7
18
LSRII (A) Trigons: filter optics and collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.8
19
LSRII (B) Blue Laser Octagon: filter optics and collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.9
20
LSRII (B) Yellow-Green Laser Octagon: filter optics and collection
pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.10
21
LSRII (B) Violet Laser Octagon: filter optics and collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.11
22
LSRII (B) Red Trigon: filter optics and collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.12
23
LSRFortessa A Blue Laser Octagon: filter optics and collection
pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.13
24
LSRFortessa A Yellow/Green Laser Octagon: filter optics and
collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.14
25
LSRFortessa A Violet Laser Octagon: filter optics and collection
pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.15
26
LSRFortessa A Trigons: filter optics and collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.16
27
LSRFortessa B and C Blue Laser Octagon: filter optics and collection
pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.17
28
LSRFortessa B and C Yellow/Green Laser Octagon: filter optics and
collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.18
29
RFortessa B and C Violet Laser Octagon: filter optics and collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.19
30
LSRFortessa B and CTrigons: filter optics and collection pathway
BP = Band Pass filter
DCLP = Dichroic Long Pass mirror
Figure 1.3.20
31
1.4 Digital Electronics
Digital flow cytometry and the LSRs
The LSRs are BD’s top of the range benchtop analyser, offering flexibility to multi-colour
applications. They are faster: the electronics are capable of digitising signals 10 million
times per second; there is increased processing capability due to reduced electronic
“dead time”. They have better resolution: improved ADC (increased bit size data) and
electronics giving better channel resolution and detection sensitivity. We can have a full
compensation matrix: inter-laser compensation is possible and can be applied or changed
on/offline. More information can be obtained and digital pulse processing of all
parameters is possible.
Digital pulse processing
In basic terms, analog signals have a continuous distribution (i.e. they can take any
value within a defined range) and digital signals are a discrete distribution (i.e. can only
have certain binned values the number of which is determined by the digitisation).
Ultimately, all cytometers are digital but they differ in where digitisation takes place. In
any case the more discrete channels that a distribution can be divided into, the better the
stored data relates to original data.
Analog: PMT  Compensation  LIN/LOG Transformation  Digitise (by ADC)
Digital:
PMT  Digitise (by ADC)  Compensation  LIN/LOG Transformation
Fig. 1.4.1 Order of data processing in analog vs. digital systems.
In the LSRs, signals from the detector (PMT) are continuously digitised by an Analog to
Digital Converter (ADC). The ADC captures a snapshot of the electric voltage from the
PMT and represents it as a digital number that can be sent to a computer. By capturing
the voltage millions of times per second (millions of time “slices”), you can get a very
good approximation to the original light signal. The ADC in the LSRs for height is 14-bit,
this gives 1014 i.e. 16,384 channels available (16, 384 “levels” that the signal may be
assigned). Each measurement represents the height of the pulse at that time. The area
ADC gives 18-bit data i.e. 1018 or 262,144 channels. If we add together all the time slices
of a pulse, we get the area, i.e. the total amount of fluorescence coming from the cell.
32
If we attach an oscilloscope to a detector on a flow cytometer, we can see the voltage
pulse that is generated:
Photon of light
Photomultiplier
Tube (PMT)
Voltage pulse
Fig. 1.4.2 Photon conversion by PMT to a voltage pulse.
We can measure the height of the total pulse by looking at the output of the ADC
(channel number read-out):
16,384
Pulse Height
Photon of light
Photomultiplier
Tube (PMT)
0
Fig. 1.4.3 PMT generated pulse: pulse height readout from ADC.
The area of the pulse is determined by adding the height values for each time slice of the
pulse (which is determined by the speed of the ADC, which is 10MHz i.e. 10 million per
second or 10 per microsecond). The area is a better representation of the total amount of
fluorescence:
Photon of light
Photomultiplier
Tube (PMT)
Pulse Area
(between 0 - 262,144)
Fig. 1.4.4 PMT generated pulse: pulse integral readout from ADC.
33
The width parameter can be thought of as being directly related to the time that a
particle of interest takes to flow past through the laser beam.
Fig. 1.4.5 Time taken to pass through laser is proportional to width parameter.
However, in the LSR, this is a calculated rather than a measured parameter. Because the
width can also be taken as the number of time slices sampled, this is calculated as
Area/Height x 64K.
Because fluorescence signals from the PMTs are digitised immediately in digital
cytometers, there is the advantage that noise introduced by other electronic circuitry is
reduced, and cytometer dead time is reduced. The signals are sampled over tiny time
slices and each given a value; digitised values are retained in buffer memory until the
area integral and pulse width measurements are completed.
The digital values are then passed to other electronics such as gating boards, before
interfacing with the computer workstation (Diva software). Note: the data is stored as
linear, if you wanted to perform log calculations, the computer “looks up” the correct
value using a “Look Up Table”. The fcs 3 files contain the uncompensated values, (but
the compensation matrix is also saved) meaning that compensation can be augmented
post collection (remember that compensation on saved analog files cannot be taken off it is possible to add compensation using analysis software but too much compensation is
unsalvageable).
The width is a processed parameter on the LSR; H and A are both measured but we
generally use the A parameter on LSR
because it represents total fluorescence coming from the cell.
34
2.1 Commonly Used Fluorochromes
Laser
488nm Blue
633nm Red
407nm Violet
355nm UV
Collection Filter
(bandpass)
Diva Parameter
Name
530/30
530/30 blue
575/26
575/26 blue
610/20
610/20 blue
660/20
660/20 blue
695/40
695/40 blue
780/60
780/60 blue
PE-Cy7
660/20
660/20 red
APC, Alexa 633, Alexa 647, TO-PRO3, Cy5, DRAQ5, DRAQ7, Dye Cycle
Ruby
730/45
730/45 red
APC-Cy5.5,
780/60
780/60 red
Fluorochromes
FITC, Alexa 488, eGFP, CFSE, Dye
Cycle Green
PE, Cy3, Sytox-Orange, Dye Cycle
Orange
PI, PE-Texas Red, PE-Alexa Fluor 610
PE-Cy5 (also “Tricolour” or
“Cychrome”), 7AAD
PerCP, PerCP-Cy5.5, DRAQ5, Dye
Cycle Ruby
APC-Cy7,
DRAQ7, Dye Cycle Ruby
DRAQ7, Alexa Fluor 750
Alexa 405, CFP, Pacific Blue, DAPI,
Hoechst (blue), Celltrace Violet, Dye
Cycle Violet, BV421, VPD450, Calcein
Violet 450, Fixable Viability Dye
eFluor 450, Cell Proliferation Dye
eFluor 450
Qdot 525, Pacific Orange, Krome
Orange
450/50
450/50 violet
530/30
530/30 violet
440/40
440/40 UV
Celltrace Violet, DAPI, Hoechst (blue)
525/50
525/50 UV
PI
Fig. 2.1.1 This table gives examples of the fluorochromes most commonly used on
LSRIIA, showing which laser they are excited by and which filter should be used to
collect the signal. All of these fluorochromes can be used with the standard LSRII A filter
arrangement as shown in section 1.3.
35
Laser
488nm
Blue
633nm
Red
407nm
Violet
561nm
YellowGreen
Collection Filter
(bandpass)
Diva Parameter
Name
530/30
530/30 blue
575/26
575/26 blue
695/40
695/40 blue
660/20
660/20 red
710/50
710/50 red
780/60
780/60 red
450/50
450/50 violet
510/20
510/20 violet
560/40
560/40 violet
Qdot 565, Pacific Orange, Krome
Orange, BV570
605/40
605/40 violet
Qdot 585, Qdot 605, BV605
660/40
660/40 violet
Qdot 655, BV650
585/15
585/15 yellow
PE, Alexa Fluor 555
620/40
620/40 yellow
660/20
660/20 yellow
PE-Texas Red, M-Cherry, M-RFPMars
PE-Cy5 (also “Tricolour” or “Cychrome”),
7AAD, E2-Crimson, M-Plum
705/70
705/70 yellow
780/60
780/60 yellow
Fluorochromes
FITC, Alexa 488, eGFP, CFSE, Dye Cycle
Green
PI, Cy3, Sytox-Orange, Dye Cycle
Orange
PerCP, PerCP-Cy5.5, PerCP-eFluor710,
DRAQ5, Dye Cycle Ruby
APC, Alexa 633, Alexa 647, TO-PRO-3,
Cy5, DRAQ5, DRAQ7, Dye Cycle Ruby
APC-Cy5.5, Alexa 700,
Cycle Ruby
DRAQ7, Dye
APC-Cy7, DRAQ7, Alexa Fluor 750
Alexa 405, CFP, Pacific Blue, DAPI,
Hoechst (blue), Celltrace Violet, Dye
Cycle Violet, BV421, VPD450, Calcein
Violet 450, Fixable Viability Dye eFluor
450, Cell Proliferation Dye eFluor 450
Qdot 525, BV510, BD Horizon V500,
DRAQ7
PE-Cy7, DRAQ7
PE-Cy5.5,
Fig. 2.1.2 This table gives examples of the fluorochromes most commonly used on
LSRIIB, showing which laser they are excited by and which filter should be used to
collect the signal. All of these fluorochromes can be used with the standard LSRIIB filter
arrangement as shown in section 1.3.
36
Laser
488nm
Blue
561nm
YellowGreen
355nm UV
633nm
Red
405nm
Violet
Collection Filter
(bandpass)
Diva Parameter
Name
530/30
530/30 blue
580/30
575/26 blue
610/20
610/20 blue
695/40
695/40 blue
PerCP, PerCP-Cy5.5, PerCP-eFluor710,
DRAQ5, Dye Cycle Ruby
780/60
780/60 blue
PE-Cy7
582/15
582/15 yellow
PE, Alexa Fluor 555
610/20
610/20 yellow
670/30
670/30 yellow
710/50
710/50 yellow
PI, PE-Texas Red, M-Cherry, M-RFPMars
PE-Cy5 (also “tricolour” or “cychrome”),
7AAD, E2-Crimson, M-Plum
PE-Cy5.5, DRAQ7
780/60
780/60 yellow
PE-Cy7,
450/50
450/50 UV
DAPI, Hoechst (blue), Celltrace Violet
530/30
530/30 UV
670/14
670/14 red
PI
APC, Alexa 633, Alexa 647, TO-PRO-3,
730/45
730/45 red
APC-Cy5.5,
780/60
780/60 red
450/50
450/50 violet
525/50
525/50 violet
APC-Cy7, Alexa Fluor 750, DRAQ7
Alexa 405, CFP, Pacific Blue, DAPI,
Hoechst (blue), Celltrace Violet, Dye
Cycle Violet, BV421, VPD450, Calcein
Violet 450, Fixable Viability Dye eFluor
450, Cell Proliferation Dye eFluor 450,
VioBlue
Qdot 525, BV510, BD Horizon V500,
Fixable Viability Dye eFluor 506,
VioGreen
Fluorochromes
FITC, Alexa 488, eGFP, CFSE, Dye Cycle
Green
PE, Cy3, Sytox-Orange, Dye Cycle
Orange
PI, PE-Texas Red
DRAQ7
Cy5, DRAQ5, DRAQ7, Dye Cycle Ruby
DRAQ7, Dye Cycle Ruby
Fig. 2.1.3 This table gives examples of the fluorochromes most commonly used on
LSRFortessa A, showing which laser they are excited by and which filter should be used
to collect the signal. All of these fluorochromes can be used with the standard
LSRFortessa A filter arrangement as shown in section 1.3.
37
Laser
488nm
Blue
561nm
YellowGreen
355nm UV
639nm
Red
405nm
Violet
Collection Filter
(bandpass)
Diva Parameter
Name
530/30
530/30 blue
710/50
710/50 blue
586/15
586/15 yellow
PE, Alexa Fluor 555
610/20
610/20 yellow
670/30
670/30 yellow
PI, PE-Texas Red, M-Cherry, M-RFPMars
PE-Cy5 (also “Tricolour” or “Cychrome”),
7AAD, E2-Crimson, M-Plum
710/50
710/50 yellow
780/60
780/60 yellow
450/50
450/50 UV
DAPI, Hoechst (blue), Celltrace Violet
530/30
530/30 UV
670/14
670/14 red
PI
APC, Alexa 633, Alexa 647, TO-PRO-3,
Cy5, DRAQ5, DRAQ7, Dye Cycle Ruby
730/45
730/45 red
APC-Cy5.5,
780/60
780/60 red
450/50
450/50 violet
525/50
525/50 violet
540/30
540/30 violet
586/15
586/15 violet
BV570
610/20
610/20 violet
BV605
670/30
670/30 violet
BV650
Fluorochromes
FITC, Alexa 488, eGFP, CFSE, Dye Cycle
Green
PerCP, PerCP-Cy5.5, PerCP-eFluor710,
DRAQ5, Dye Cycle Ruby
DRAQ7
PE-Cy7, DRAQ7
PE-Cy5.5,
DRAQ7, Dye Cycle Ruby,
APC-Cy7, Alexa Fluor 750, DRAQ7, Fixable Viability Dye eFluor 780
Alexa 405, CFP, Pacific Blue, DAPI,
Hoechst (blue), Celltrace Violet, Dye
Cycle Violet, BV421, VPD450, Calcein
Violet 450, Fixable Viability Dye eFluor
450, Cell Proliferation Dye eFluor 450,
VioBlue
Qdot 525, BV510, BD Horizon V500,
Fixable Viability Dye eFluor 506,
VioGreen
Fig. 2.1.4 This table gives examples of the fluorochromes most commonly used on
LSRFortessa B and C, showing which laser they are excited by and which filter should
be used to collect the signal. All of these fluorochromes can be used with the standard
LSRFortessa B filter arrangement as shown in section 1.3.
38
*Which LSR? *
The configurations of the four LSRs are different (see section 1.3); this may mean
that one machine is more appropriate than the other for a specific experiment.
These applications can only be run on a specific LSR:
-LSRIIA or LSRFortessas: Hoechst side population and Indo-1 calcium flux.
-LSRIIB or LSRFortessas: RFP and variants.
These applications may be better using the yellow laser on LSRB or LSRFortessas:
-Any experiment using PE and PE tandems (there is less autofluorescence and
better S/N ratio).
-Experiments combining GFP or CFSE with PE.
Note: For PerCP or PerCP variants (PerCP-Cy5.5 for example), use 488 (blue)
laser excitation NOT the 561nm (yellow).
2.2 Fluorochrome Selection
There are many factors that will affect the choice of fluorochromes for an experiment.
Please speak to a member of the FACS Lab when designing a new experiment and
before purchasing expensive reagents!
Viability dyes
The presence of dead and dying cells in a sample can have a detrimental effect on the
analysis and interpretation of fluorescence data. Dead cells may lose markers present
on/in live cells and can become autofluorescent, i.e. giving a positive fluorescent signal
although no fluorochrome is present. They may also non-specifically take up antibodies,
generating artefacts that are difficult to exclude when you wish to analyse your data.
These problems are easily overcome by using a viability dye, sometimes referred to as a
live/dead dye. These dyes are excluded by the intact membranes of live cells, but can
enter dead and dying cells. Therefore, gating on the population negative for the viability
dye easily excludes dead cells.
39
Dye
Propidium iodide (PI)
7AAD
TO-PRO-3
Excited by:
Blue Laser/Yellow laser
Blue Laser /Yellow laser
Red Laser
Collected in (LSRIIA/B - LSRFortessa A/B):
610/20bp / 575/26bp
660/20bp / 610/20bp
660/20bp
DAPI
DRAQ7
UV laser / Violet laser
Red laser
440/40bp / 450/40bp / 450/50bp
670/14bp / 660/20bp / 710/50 bp / 730/45bp
/780/60bp
Fig. 2.2: Examples of commonly used viability dyes.
*Tips and Tricks*
The dyes mentioned above can only be used in unfixed samples. In fixed samples the
dye would enter all cells. The most popular viability dye for the LSRs is DAPI. This is
because it is excited by the violet and UV lasers and rarely overlaps with another
fluorochrome in the same experiment.
Fixable LIVE/DEAD amine reactive dyes
If you wish to be able to fix your sample, you may try using the amine-reactive dyes
from Molecular Probes, Invitrogen. In membrane-compromised cells, the dye has access
to free amines both in the cell interior and on the cell surface; in viable cells, the dye can
react only with cell-surface amines, generating lower levels of fluorescence. Up to 50fold difference in intensity can be obtained between live and dead cells, and the staining
is preserved following formaldehyde fixation.
Availability
This may be the major factor limiting your choice of fluorochrome combination. Although
a selection of different directly conjugated fluorochromes are available for commonly
used antibodies such as CD4, this may not be the case with less common antibodies. In
many cases it is necessary to use a two-step staining process involving primary and
secondary antibodies and in this case great care must be taken to avoid non-specific
staining and cross-reaction between antibodies. Please ask a member of the FACS Lab if
you need further advice on this.
40
Physical Characteristics
Physical characteristics of fluorochromes may influence their selection, for example, the
size of different fluorochromes varies considerably and when doing intracellular staining
it may be necessary to select a smaller fluorochrome. The brightness (quantum
efficiency) of different fluorochromes also varies. For multi-colour staining it is advisable
to use the brightest fluorochromes to identify the dimmest or least expressed markers.
2.3 Combining fluorochromes
Spectral overlap
With
all
flow
cytometers,
problems
can
be
encountered
when
using
multiple
fluorochromes because the emission of each fluorochrome can “spill over” into the
detectors of others. This problem increases with the number of fluorochromes used and
is therefore especially relevant to the LSRs.
Fig. 2.3.1 Emission spectrum of two commonly used fluorochromes. FITC (green line) is
detected with a 530/30 filter and PE (purple line) is detected with a 575/26 filter. These
filters are chosen to capture the maximum emission of their respective fluorochromes.
However, some FITC emission can be seen spilling over into the 575/26 spectral region
and dim PE emission can be seen in the 530/30 and 660/20 (PE-Cy5) regions.
Problem pairs
Because of the spectral overlap, certain combinations of fluorochromes can be
problematic, in many cases this will depend on how bright the fluorochromes are. For
example, combining a very bright GFP with dim PE could be difficult, but a dimmer GFP
41
signal may not cause problems. Other combinations will always be potentially difficult,
an example of this is given in Fig. 2.3.2 below.
Fig. 2.3.2 Spectral overlap of PE-Cy5 (excitation dark green line, emission light green
line) and APC (excitation dark purple line, emission light purple line). PE-Cy5 is optimally
excited by the 488 laser and APC by the red laser, however some excitation of the Cy5
component of PE-Cy5 can occur with the red laser. Both fluorochromes emit in the
660/20nm region so it can be very difficult to separate the red excited PE-Cy5 signal
from the APC; take care in checking the voltages for APC and PE-Cy5 to obtain a good
balance of signals and still be able to compensate PE-Cy5 out of the APC channel.
Example combinations
Fig. 2.3.3 2-colour staining, using FITC (blue line) and APC (purple line). The emission of
these two fluorochromes is well separated resulting in very little spectral overlap. If
using live (unfixed) cells DAPI should be added to this combination as a viability dye.
42
Fig. 2.3.4 3-colour staining, using FITC, APC and PE-Cy7 (yellow line). The emission of
PE-Cy7 is higher than that of both FITC and APC so there is little spectral overlap. If
using live (unfixed) cells DAPI should be added to this combination as a viability dye.
*Useful websites: *
The following websites all enable you to view the excitation and emission of various
fluorochromes and compare different fluorochromes to determine the likelihood and
severity of spectral overlap.
http://www.bdbiosciences.com/spectra/
http://probes.invitrogen.com/resources/spectraviewer/
http://www.ebioscience.com/resources/fluorplan-spectra-viewer.htm
43
2.4 Compensation
Compensation is used to control the effects of spectral overlap. The aim of compensation
is to account for spectral overlap of fluorochromes into adjacent detectors. This is very
important for the reasons explained in section 2.3 above. The LSR has a number of
features that help to improve the ease and accuracy of compensation. Having the correct
controls
is
also
very
important.
For
further
explanation
on
the
principles
of
Compensation, please speak to any member of the FACS Lab.
Controls
For each of the fluorochromes used in the test samples, a single colour positive control is
needed for compensation.
Each control should ideally be made up of a population of negative or unstained cells and
a population of single colour positive cells. Both the positive and negative cells should
have the same level of background autofluorescence, ideally this will mean ensuring that
they are the same type of cell, e.g. lymphocytes.
Ideally the positive and negative populations in the controls should both be large, well
defined and well separated. However, if you are looking for a rare population or have a
very limited number of cells this might not be possible. There are three ways to
overcome this problem:
1.
Use different cells for your compensation controls - e.g. if the test samples are
primary tissue in limited supply, find a cell line that expresses the markers of
interest and use these for the compensation.
2.
Use a different antibody conjugated to the same fluorochrome – if a marker
of interest is rare or possibly absent in the control cells, it is better to use a different
antibody, directed against a more common marker, but carrying the same
fluorochrome as will be used in the test. CD45 antibodies are often used for this
purpose when studying rare lymphocyte markers. It is important to note however
that the exact same fluorochrome should be used, e.g. GFP and FITC may both emit
a signal detected in the 530/30 channel, but they are not the same fluorochrome.
Tandems are trickier due to variations in chemical conjugation: if a tandem
conjugate is being used, e.g. PE-Cy5, the same batch of tandem should be used.
44
However, it is safer still to use a cell line highly expressing the marker and label with
the same antibody you are planning to use.
3.
Use compensation beads: If the marker of interest is rare or absent on/in the
control cells, you can still use the antibody if you substitute the cells for “comp
beads”. Comp beads are polystyrene microparticles, available as Anti-Rat or Antimouse Ig, kappa. They bind to rat or mouse light chain Ig antibodies (check which
ones are right for your antibodies). Beads with no binding capacity are also available
and can be used as a negative control to determine background fluorescence,
ensuring that clear positive and negative populations are present in all compensation
controls.
Compensation Beads
To prepare the controls the antibodies to be used are incubated with the beads as
follows:
Combine 100µl staining buffer + one drop negative beads + one drop of Comp
beads (Anti-mouse or Anti-rat) + antibody (at the concentration used in the
experiment).
Incubate for 30min (RT in the dark).
Wash with 2ml of staining buffer and resuspend compensation controls in 500µl
staining buffer and run them on the flow cytometer.
Cells can also be added to the compensation control tubes to check for
autofluorescence from your cells and to help establish your baseline voltages.
A
B
Fig. 2.4.1 A Scatter plot showing a large scatter gate that includes the Comp beads and
cells. B APC-Alexa750+ve beads can clearly be distinguished from unstained particles
and used for compensation (plot showing only scatter gated events).
45
The Bi-exponential Display
The bi-exponential display is a feature of digital cytometers. It allows you to see below 0
on a log axis. This means that any events that would normally appear crushed on the
axis will be visible; the bi-exponential display does not change the data, it merely
transforms it to present it in a visually understandable way. This makes it much easier to
do compensation, and is particularly useful for seeing if a sample has been over
compensated, as shown below.
A
C
B
D
E
Fig. 2.4.2 A shows an uncompensated sample on the standard LSR 5 log display, B
shows the same uncompensated sample viewed using the bi-exponential display. C
shows the sample once compensation has been applied “by eye” without using the biexponential display, the centre of the positive population appears to be roughly in line
with the centre of the negative population. D shows what the compensation used in C
looks like when viewed with the bi-exponential display. It can clearly be seen that the
sample is actually over compensated, with the positive events below the negatives. E
shows correct compensation, as viewed on the bi-exponential display.
46
The bi-exponential display function can be selected for individual axes and plots. To
switch it on: select the plot, then in the Instrument window check the boxes for the
relevant axes.
Note: The scale of the axis will automatically adjust to give the best display of all current
events. During sample acquisition this can lead to large blank areas appearing in the
negative region, however this should be corrected when all the collected data is
displayed for analysis. You may find it best to turn off the bi-exponential display when
acquiring data.
Compensation – median fluorescence technique
It is recommended that you use the statistics functions in Diva to calculate the exact
amount of compensation required. This is done by drawing gates around the positive and
negative populations and comparing the median fluorescence of the two. For example, if
you are compensating FITC out of PE, the median fluorescence of the double negative
+
-
population will be X. The FITC PE cells have no PE and should therefore give the same
median PE signal as the double negative (X). However, because of the spectral overlap
+
-
of the FITC signal into the PE channel, the FITC PE population has an increased median
PE fluorescence of Y. To correctly compensate this sample you simply increase the
amount of compensation applied until Y=X. You just have to imagine a line in the centre
of the positive population that needs to be lined up with the centre of the negative
population. You will be taken through the process of compensation in section 3.
*Tips and Tricks*
If you are using any fluorochrome pairs that could be difficult to compensate, e.g. PECy5 with APC, it is a good idea to run these first and do a rough check on the
compensation by eye before saving all the samples. This prevents you from having to
re-record all your controls if you need to change the voltages on one to help
compensation. After checking the compensation reset it to zero before recording the
sample.
47
Copying the spectral overlap
Once you have calculated your compensation you will need to apply it to all future tubes.
This can be done by just writing down the compensation applied to each tube and typing
it in for the sample tube, or by using the copy spectral overlap function. For the latter,
once you have set the compensation for a control, expand the tube and right click on
instrument settings. Select “Copy spectral overlap” then right click on the instrument
settings for the experiment and select “Paste spectral overlap”. Repeat this procedure
after compensating each control. Having completed the compensation, click Next; the
new tube and all subsequent tubes should contain all the compensation and be ready for
you to run your samples. There is an example experiment in section 3 where you can try
this out.
Automatic compensation
As the number of fluorescence parameters in an experiment increases, compensation
becomes increasingly difficult to set manually. For a six-colour experiment, 30 spectral
overlap values need to be adjusted, and for an eight-colour experiment, 56 values need
to be adjusted. The process of manually correcting spectral overlap values can take
several hours and is very difficult to set accurately. The Compensation Setup feature in
BD FACSDiva software is designed to automatically calculate spectral overlap values for
an experiment, saving time and eliminating the inaccuracies introduced with manual
compensation.
We
recommend
that
for
any
experiment
with
three
or
more
fluorochromes compensation should be set using the automatic feature.
Compensation Setup is designed to work with single-stained controls. These controls can
consist of single-stained cells or capture beads. An unstained control is required as well,
in a separate tube or in the same tube as the single-stained controls. As mentioned, you
can find more details about the process of compensation in section 3.
48
We will now run an example experiment with cells, FITC and PE beads.
Open the software (double click the FACSDiva icon on the desk top).
There is no password, just hit “Enter” at the password window.
*Checklist before starting experiment*
Has the LSR been switched on long enough for the lasers to warm up?
Is the sheath tank full and waste tank empty?
Is the standard configuration of optical filters in place?
Is it clean (who used it last? when was it used last?)
- Worth giving a few Primes, checking cleanliness with dH20
3.1 The Browser, Worksheet, Inspector and
Acquisition windows
1. Browser
2. Cytometer
5. Worksheet
4. Acquisition Dashboard
3. Inspector
Fig. 3.1.1 Diva v6 software windows
50
1. The Browser: where you can make new folders
and experiments, and access or delete acquired
experimental data.
Fig. 3.1.2 The Browser window
2. Cytometer: controls all instrument related electronic functions. This window cannot be
accessed unless an experiment is open, and the arrow to the left of a tube in the browser is
selected (green). Instrument connection status is shown in the footer of the window. There
are a number of tabs below the header including Parameters (equivalent to Detectors and
Amps in CellQuest) and Compensation; these will be discussed further in later sections.
3. Inspector: Each Diva window has information that can be edited/changed via the
“Inspector”, which is accessed by making a window active.
4. Acquisition Dashboard: is the main panel for collecting data. Here you can start
acquiring/recording data, and add storage and stopping gates for acquisition. Changes to
the number of events displayed and recorded can also be made here. Data recording
progress is shown as a coloured bar; when completed, click the NEXT TUBE icon to move
on to the next sample. The sample flow rate (events/sec), aborted events and time elapsed
is also shown.
5. The Worksheet: where you can make plots and histograms. There are two types of
worksheets but we only use the Global type worksheet to acquire data. Data analysis is
performed using FlowJo.
51
*Trouble-shooting: Connectivity Problems*
The Instrument/Cytometer window displays LSR instrument connection status to the
workstation software. If it seems to have trouble connecting (takes too long, or shows
“Instrument Disconnected”) try re-starting the computer.
If this doesn’t help, try switching off BOTH the computer and LSR, then switching back on.
3.2 Example experiment
The rest of this section will take you through the basics of using Diva by
running an example experiment using cells and fluorescent beads.
52
Sample tubes:
*DAPI will be included in all tubes as a viability dye*
Tube 1: unlabelled cells + unlabelled beads (Unlabelled control)
Tube 2: unlabelled cells + FITC beads (FITC control)
Tube 3: unlabelled cells + PE beads (PE control)
Tube 4: unlabelled cells and both beads (the sample)
Left Click on
Administrator at the top of
the tree of folders (to
highlight the location).
Left Click on the folder
icon to create a new
folder. Re-name the new
folder with your NAME (it
should appear at the
bottom of the tree under
Administrator).
Click
on
the
New
Experiment icon (brown
book) to make a new
experiment for today.
Click
on
the
New
Specimen icon (syringe)
to create a new specimen.
Rename the specimen as
Cells+Beads.
Rename the first tubeUnlabelled control.
Highlight today’s
experiment; in the
Inspector window select
Experiment tab; check 5log axis box.
Highlight Tube_001:
Instrument window:
Select the Parameters tab
We will be using FSC,
SSC, 530/30blue (FITC),
575/25blue
(PE)
and
440/40violet
(DAPI).
Delete
the
other
parameters.
*Renaming things*: often done by right clicking
on the item to access a menu.
TIP: Include as much information as possible in
your experiment (i.e. date, reagents, tube names
etc.) as this is saved in the FCS files and is
accessible during analysis.
The new folder should appear under (in) the folder with
your name; re-name today’s folder with your INITIALS,
DATE, and LSRIIA, LSRIIB or FORTESSA (e.g.
MW010212lSRIIA).
If you are using two cell lines, you could make a
separate specimen for the second, or if you are looking
at samples from several patients/animals each could be
a specimen. Give the specimen a short name.
If you expand the browser at the level of specimen
(click on “+” sign, left of the specimen) you will see a
tube icon Tube_001; you may rename this if you wish.
The Inspector window contains a number of tabs for a
variety of functions, some useful (including re-labelling
parameters with names), and some redundant (e.g.
acquisition and storage information - which can be
changed through the acquisition controls).
The Instrument window- further considerations
Minimising file size: Data files can be large, so
one important thing to remember to do before
saving data is to delete any unused
parameters/colours (click the bullet on the left
of the parameter name and click the Delete
button on the bottom left) - you can select
several by clicking the first one, holding shift and
clicking the bottom bullet point.
Owing to a bug in Diva: it is necessary to leave
at least two parameters from the blue laser in
the Instrument window.
53
Instrument Window:
In the Parameters Tab set
parameters to log or linear
as appropriate.
Instrument Window:
In the Parameters tab: Tick
the W box for both FSC and
SSC to permit doublet
discrimination.
Instrument Window:
Threshold tab: set FSC and
SSC to 5000 (we will be
keeping it low since we are
running beads as well as
cells).
Inspector Window
(highlight Experiment)
Label
the
fluorescence
channels
with
the
fluorochromes
you
are
using.
Draw the following plots
and histograms.
Plot 1: FSC-A vs. DAPI
Plot 2: FSC-A vs. SSC-A
Plot 3 & 4: FSC-A vs. SSCW and/or FSC-A vs. FSC-W
Plot 5: FITC vs. PE
Histogram1:FITC
Histogram 2: PE
Under the Parameters tab: generally, FSC/SSC will be
linear and fluorescence will be log.
Digital data are stored as linear so you can
actually change between LOG/LIN after
acquisition (however, if you wish to do this you
must ensure that all events are on scale for both
log and lin displays because you can’t adjust the
voltages after acquisition).
Exceptions to using LOG for fluorescence
include DNA and other measurements where
differences between +/- are quite small; when
doing BrdU analysis it is also sometimes worth
trying a linear scale for better resolution.
Doublet Discrimination: A, H and W can be
measured for all parameters. In general, use A as it
represents the total fluorescence. W is analogous to
time spent passing through the laser beam so we can
use this to discriminate against cells stuck together. W
from either scatter parameter can be used (or both if
cells are sticky and extra stringency is required).
Doublet discrimination is particularly important in DNA
analysis where a different strategy should be used;
please see a member of staff about this.
Threshold values: These are channel number values
used to tell the computer what to ignore as
noise/debris (i.e. events below a certain channel
number will not be displayed or recorded). We tend to
use both FSC and SSC. In analysis these can be set
relatively high (e.g. 10-15K).
Use of threshold aids processing time as fewer
events are analysed. For samples with a lot of debris
this is particularly useful.
In the Inspector window, labels tab:
530/30 blue: label with FITC
575/25 blue: PE
440/40 UV: DAPI
Plots: Using the dot plot icon, drag and let go, or click
on the workspace to get a standard size plot.
Parameters displayed can be changed by clicking on
the name of the parameter in the plot axis (drop down
menu).
54
Working with Plots, Regions and Gates
There are 3 types of plots available on the LSR, the icons used to select these are circled in
red below. These will allow you to draw dot plots, contour plots and histograms
respectively.
Fig. 3.2.1 Icons at top of the worksheet window used for drawing plots (red) and regions
(green). Plot drawing icons (left to right) Dot plot, Contour plot, Histogram. Gate Drawing
icons (left to right): Autopolygon, Snap-To gate, polygon, Rectangle, Quadrant, Interval,
Autointerval, Snap-To interval.
Plots
Dot plots and contour plots are both 2 dimensional displays allowing you to compare 2
different parameters on the same events. For example, this will allow you to see which
FITC positive events are also APC positive. An example of a dot plot is shown in fig.
3.2.3.B. Dot plots represent each event as a dot, where a population of events all have
similar staining they will form a distinct cluster of dots.
A contour plot shows the population as a set of contour lines. Lines are drawn as a
percentage of the total events; the area between each line has the same percentage of the
total events so that the bigger the population is, the closer together the lines will be. This is
particularly useful to visualize the distributions of populations that are very close together,
or overlapping.
55
A1
A2
B1
B2
Figure 3.2.2 A1 shows a sample where two populations (P2 and P3) of interest are identified
by drawing gates on a dot plot. In this case, the gates are drawn so that they are touching.
A2 shows the same data presented on a contour plot: it can be observed that there is likely
to be slight overlap of P2 and P3. When the gates are re-drawn with some separation, as
shown in B1, the separation of P2 and P3 is increased.
Histograms are single parameter presentations of data and should be used with careful
consideration as they have some significant limitations, meaning that they are not suitable
for all types of experiments. One commonly observed error is using histograms to gate
positive
events
for
analysis
whilst
not
taking
into
account
the
contribution
of
autofluorescence to the signal. This is particularly common for people doing single colour
experiments such as FITC and PE, as cells often have “greenish” autofluorescence. Fig.
3.2.3 demonstrates clearly the danger in gating off a histogram.
56
A
B
Fig. 3.2.3 A common error of using a histogram instead of a 2-parameter plot to observe
GFP: A shows an interval region on a histogram used to select events that are GFP+ve,
which seems to be around 18% of the total events. B is the same population of cells shown
on a 2-parameter plot, with regions drawn to show that, of the 18% selected by the
interval region in A, around 15% are autofluorescent, and only 2.5% are truly GFP+ve.
Regions and Gates
To select a population of cells for further analysis or to obtain statistics on the population a
“region” can be drawn around it. There are a number of tools for drawing regions, the icons
for these were shown in fig. 3.2.1.
Region Name
Used on plot
Use:
type(s)
Autopolygon
Dot/contour
To automatically draw a polygon around a population.
Snap-To gate
Dot/contour
To automatically draw a polygon a population. The
shape of the polygon will adjust as the population
changes.
Polygon
Dot/contour
To
manually
draw
a
polygon
region
around
a
population.
Rectangle
Dot/contour
To manually draw a rectangle or square region
around a population.
Quadrant
Dot/contour
To divide the plot into “quarters” (not necessarily of
equal size), each of which is a region. Arms may be
hinged (so that not 90 degrees).
Interval
Histogram
To manually select a region on a histogram.
Autointerval
Histogram
To automatically select a region on a histogram.
Snap-To interval
Histogram
To automatically select a region on a histogram, the
region will adjust as the population changes.
Fig. 3.2.4 Types of regions/gating tools available for specifying populations of interest.
57
Combining Regions
Once you have drawn a region it will be given a name, e.g. P1 (meaning population 1), you
can then select to show only the cells within this region on a new plot. To do this right click
on the new plot and select Show Populations→P1.
If you draw a new region on the new P1 gated plot, this region will become P2 and will be a
“daughter” of P1 (the parent). Regions drawn on the same plot will be siblings and not
linked to each other. You can view the organisation of your regions by opening the
population Hierarchy window. Do this by right clicking on the margin of any plot and
selecting view → population hierarchy.
The word “gate” is often used to describe a region. However, a gate can also be a
combination of parent and daughter regions and refers to which events would be displayed
on a plot gated on Pn (when Show Populations→Pn was used). Where a population has no
parent (e.g. P1) the single region defining it can be referred to as a gate because every
event within it would be displayed. Where the region has a parent (e.g.P2) only cells that
fell within the daughter region and all parent regions would be displayed.
*More Points To consider about gating*
Regions drawn on negative populations (excluding scatter and viability gates)
need to be pushed to the very edge or slightly off the axis to avoid excluding
negative events that are crushed up against the axis.
Drawing regions very close together may result in unexpectedly overlapping
populations even if the regions themselves were not overlapping. This is caused
by slight statistical variations in the positioning of the events on the plot. It is
useful to obtain a general picture of how well separated the populations are by
looking at a contour plot
Data follows a biological distribution that does not always fit with rigid gating.
Be consistent and logical in how you draw regions, to minimise subjective bias
for inter-experiment comparison.
Quadrants/ rectangles may not necessarily fit plots with four compensated
populations (-/-, +/-, -/+ and +/+), particularly when there is a range of
antigen expression involved, using these straight line regions can mean part of
a population ends up in the wrong region. It’s worth considering using a
polygon or altering the quadrant so that the sections are different sizes/shapes
(this is thoroughly discussed in the Data Analysis section of Howard Shapiro’s
Practical Flow Cytometry 4th Ed.
58
On LSR: hit RUN in LO
Remove tube of H20
Install Tube 1 onto SIP
(UNLABELLED CONTROL).
*TIP*
Quickly CHECK your sample before running: Flick
to mix, make sure NO CLUMPS ARE VISIBLE!
Pipette if necessary.
Acquisition Controls:
Click Acquire.
Show 5000 events on plots.
*TIP*
Monitor the threshold rate in the Acquisition
Status window to be sure that your sample is
running and hasn’t clogged.
Adjust scatter PMTs to
see both cells and bead
population on the scatter
plot.
Scatter: We want to be able to see the whole
population of interest on our scatter plot, such that we
don’t lose the edges, and so that we can just see the
debris, keeping it in the bottom end of the scale.
Where this is actually placed is subjective, and
dependent on what you are doing. For samples where
several cell populations are present such as in whole
blood, if you were interested in looking at lymphocytes,
you may choose to increase scatter to bring these out
further on the plot, but this will push the granulocytes
off. If you were interested in apoptosis, you may
choose to increase scatter to expand the area of
interest- where the apoptotic bodies with lower FSC
may fall.
Select a gating tool:
Draw a gate (P1) around
the
DAPI
negative
populations.
This
will
include cells and beads.
Population
Hierarchy:
Open by right clicking on
any plot and selecting show
population hierarchy.
Right click on the scatter
plot-->Show Population P1.
Select Gating tool: Draw
a large second gate (P2) to
include the cells and beads.
Show P2 on the FSC-A vs.
SSC-W plot and draw a
gate (P3) around the tight
singlet population of lowest
SSC-W.
Show P3 on the rest of the
plots and histograms.
At this point we can start running the samples and
adjusting the voltages for the detectors. Remove the
tube of water and place the machine into RUN. Make
sure the speed is LO. Under Acquisition Controls choose
to display 5000 events (so the plots refresh quickly).
The scatter plot will now only display events from P1.
Notice that the software organises this second gate as
a daughter of P1 because P1 was shown first on the
plot before P2 was drawn.
Notice that the singlet population is tighter on the
FSC-A vs. SSC-W than vs. FSC-W. As mentioned
previously, sometimes even after gating singlets
on FSC-A vs. SSC-W there are still some clumps
present that can be seen on the FSC-A vs. FSC-W
plot.
59
Fig. 3.2.5 An example of the FACSDiva Worksheet showing the plots created in the
example experiment. This is data generated by running the unlabelled control.
60
Once the scatter looks ok and the basic gates have been drawn, the fluorescence plots will
look cleaner due to the exclusion of dead and dying cells, and we can adjust the PMT
voltages.
10 5
10
<780/60 blue 5-A>: PE-Cy7
<780/60 blue 5-A>: PE-Cy7
10 5
10
4
27.2
10
10
37.8
10
3
10
2
0
4
3
2
0
0.83
0
A
2
3
0.53
8.55
26.5
4
10
10
10
<675/20 blue 4-A>: PerCP
2
0
5
10
Ungated
B
4000
10
3000
SSC-A
95.1
2000
3
2000
1000
48.5
2
FSC-W
<450/40 violet1-A>: DAPI
10
5
10
4000
3000
4
4
Singlets
10 5
10
3
10
10
10
<675/20 blue 4-A>: PerCP
1000
99.2
0
0
0
C
1000
Ungated
2000
FSC-A
3000
4000
0
1000
D Live
2000
FSC-A
3000
4000
E
0
0
1000
2000
FSC-A
3000
4000
Scatter
Fig. 3.2.6 The importance of correct gating: A shows fluorescence data from all events
acquired in a sample from a two-colour experiment. B shows the fluorescence of the same
sample following gating to exclude events that are dead cells and debris. The gating
strategy was as follows: the DAPI negative population was selected first (C); this live
population was then shown on a scatter plot (D) and gated to exclude debris and obvious
clumps. The live+scatter gated events were then further gated to exclude doublets by
excluding events with a high FSC-W signal (E). The difference between the two plots is
very clear especially when looking at the double negative population.
61
Baseline PMT Voltages
The optimum voltage set up is one where the signal from positively stained populations
can be best separated from negative/unstained populations, with negative populations
being positioned above the electronic noise and at the same time allowing dim populations
to be distinguishable above background.
BD published an Application Note (Establishing Optimum Baseline PMT Gains to Maximise
Resolution on BD Biosciences Digital Flow Cytometers) which can be found on their website.
This protocol basically involves running fluorescent beads and plotting %CV (a measure of
peak distribution defined as standard deviation of the peak divided by the mean channel
number of the peak, times 100, reflecting how “fat” a peak is) at different PMT voltages to
find the minimum voltage where fluorescence %CVs are at their tightest (values start to
plateau) and hence the minimum voltage for optimal resolution and sensitivity.
http://www.bdbiosciences.com/pharmingen/products/display_product.php?keyID=126
In practice, one finds that when voltages are set such that positive signals and negative
signals are separated and voltages are balanced against other fluorochromes used (so that
spill-over can be compensated out) they will be above the baseline anyway.
62
Compensation
UNLABELLED CONTROL:
In general as we go up the spectrum to the red end
we
need
to
increase
the
voltage
(fewer
photoelectrons produced so need to amplify more)
Adjust
PMT
voltages:
Place the signal from the
negative cells in the first
two logs of your plots.
Click Restart in the Acquisition controls. Check that
the separation for PE is the best it can be (play with
voltage).
Run PE control tube:
Check fluorescence for PE.
Do the same with FITC
control but ensure FITC
voltage> PE voltage.
*TIPS AND TRICKS*
By convention, we are used to running controls
starting with the shortest emission wavelength
fluorochrome (‘greenish’). It is actually often
more efficient to start at the other end- with the
‘far red’, long wavelength fluorochromes. These
have the least energy and often require a higher
PMT voltage. Once these are set (+ve’s and –
ve’s as well separated as possible), then you can
work your way back towards FITC. Working the
other way you will often find you have to readjust FITC, PE etc.
FITC +ve
PE+ve
Fig. 3.2.7 The PE control is shown on the left, on a FITC vs. PE plot. In this particular
example very little compensation will be needed to remove PE out of FITC. To the right is
the FITC control on bi-exponential display; considerable spill over into the PE channel can
be seen.
63
Acquisition Controls:
Set a Stopping Gate on P3
for data acquisition.
Record 20K events of the
unlabelled control.
Click Next icon for a new
tube; re-label as FITC
control. Record 20K events.
Repeat for PE.
Recording data: Once you have checked that the
single colour controls look ok, you are ready to record
data. It is a good idea to set a Stopping gate to stop
acquisition when the desired number of relevant
events has been seen. The most basic level is to set
this to P3 (for live, single cells).
Remember if you have a small volume to not
let the machine run out of sample/fluid.
This will introduce air into the system (e.g. if
you are running near to the bottom of your
sample, you need to be QUICK in hitting
Record to save, and getting that tube OFF.
Ideally, single colour controls for Fluorescence
Compensation have both a negative and a bright
positive population, which are well separated.
How many cells do I need to record?
To be able to draw any statistically significant
conclusions, we need to look at the actual final
population of interest. In experiments such as
immuno-phenotyping, the final population may
be very rare and the last in line of a number of
hierarchically structured gates. Rare events
follow a Poisson distribution, so we use the
following formula to calculate CV:
CV= (1/√n) x 100
(CV = standard deviation, n = cell number)
We can see that for a CV<10 (ideally CV=1)
we need at least 10K cells.
This is especially relevant with rare populations
and it is the actual number of these that is
important – it should be at least 100 relevant
events (i.e. after correcting for background).
This can be tricky as in theory both control and
test samples should be the same size!
Right click on any plot and
open the Statistics Window.
Right click on the statistics view itself to edit
information displayed in three different tabs, Header
(information about the experiment to be displayedmost of which can be un-ticked), Population (gates
can be de-selected if statistics not needed) and
Statistics (a table of all parameters with possible
statistics which can be selected for display).
64
Edit the Statistics window.
Load the data from the
FITC control by selecting
the arrow to the left of the
tube.
Draw gates around the
positive and negative
populations.
Select a fluorescence
plot and turn on the biexponential display; this
is the plot we will use for
compensation.
Compensate the FITC
out of the PE channel.
Remove all the options under the Header tab. Under
the populations tab show only P4 and P5. Under the
Statistics tab tick the box for Median and remove any
other statistics.
On a 2-parameter plot of FITC vs. PE, gate both the
negative (this will be P4) and positive (this will be P5)
populations. The FITC+ population will also appear to
be slightly PE+, i.e. curving upwards away from the xaxis.
Turn on bi-exponential display (by left clicking on the
plot once). In the Inspector window check the boxes
to show bi-exponential display for both x and y-axes.
First, we want to remove the percentage of FITC
signal that is leaking into PE channel, i.e. PE- %FITC.
On the LSR this will be 575/25blue -530/30blue.
The Compensation tab can be found in the
Instrument window. The Compensation Matrix
allows full, inter-laser compensation of all parameters
against each other. The matrix is set up with the first
fluorochrome (shortest wavelength PMT, equivalent
to “FL1”) to be subtracted from all the other
parameters, then the second and so forth down the
column. Use of the bi-exponential display while
adjusting compensation is advised to properly
observe compensated parameters.
Compensation can be added by typing a value or
using the slide bar in the right column until the single
colour control no longer appears positive in the
second channel (median fluorescence values of the
positive and negative populations are equal).
*Tips and Tricks*
When compensating a single colour control one
can think: What is in the tube is in the middle
column, and the channels that you are
removing that fluorochrome from are in the left
column.
65
Fig 3.2.8 The workspace above shows FITC compensated out of the PE channel (575/26blue
– 530/30blue) using median values. 16.7% compensation is required to bring the median
PE fluorescence of the FITC population (P5) to match those of the negatives (P4).
66
Right click on the FITC
tube. Select Copy spectral
overlap.
Right click on the PE tube.
Select Paste with zeros.
Load up the next
control: PE control.
Compensate PE out of
FITC.
On the fluorescence compensation plot, place PE on
the x axis, and FITC on the y-axis.
If we were using more colours (not counting DAPI as
it does not leak into FITC or PE channel and we are
selecting cells which are DAPI negative), one would
change the y-axis parameter to the next channel and
repeat the process of finding the medians etc, for all
the other colours.
*Putting the control colour on the x-axis and
changing the y-axis as you compensate x out is
methodical but everyone has different ways of
doing this, you may find a way that you prefer
instead.
When
all
controls
have
been
systematically
compensated and the spectral overlap copied and
pasted, the bi-exponential display can be switched off
(not critical but helps with processing if you are
collecting huge numbers of cells).
Draw gates for the FITC
and PE single positive
populations.
Using the PE control data, draw the FITC+ gate; then
use the FITC control to draw the PE+ gate. For more
information on this see the section on FMOs in the
fluorochromes section.
Click Next, and re-label
the new tube.
Apply compensation and
record sample.
Gate the cell population.
Show that these are FITC and PE negative. Also gate
the smaller bead population. Show these are FITC+
and PE+
67
Automatic Compensation
Compensation cannot be calculated if PMT voltage settings are changed while
recording compensation tubes. All tubes must be recorded with the same PMT
voltages.
The optimum voltage set up should be made before creating the compensation
controls. To do that, you should create some dot-plots on the global sheet that allow
you to see the spillover between the detectors. If you need to re-adjust the PMT
voltage after creating the compensation controls you will need to record all
compensation tubes again.
Create the compensation controls
Go to Experiment > Compensation Setup > Create Compensation Controls
A list of the parameters that will be
used as compensation controls is
created (Fig. 3.2.9). Check if you
have in the list all the desired
parameters (use options Add or
Delete if necessary)
Fig. 3.2.9
Leave the Include separate unstained control tube/well checkbox selected
(highlighted with the red box) when you are running unstained sample as one of your
compensation controls. Deselect the checkbox when you are including unstained
sample in each of your stained control tubes or wells.
When you click OK, a new specimen named
Compensation
added
to
Browser,
Controls
(Fig.
3.2.10)
the
open
experiment
with
tubes
for
each
in
is
the
specified
parameter. A normal worksheet is created
for each single colour control, along with an
Fig. 3.2.10
unstained control worksheet if the checkbox was selected.
The experiment’s cytometer settings are copied to the controls, all
compensation values are set to zero, and the Enable Compensation checkbox
is deselected.
68
Install the first stained tube onto the cytometer and start acquisition. Adjust the
pre-defined P1 gate to your population (Fig. 3.2.11) and press record. When
recording is finished install the next stained control tube onto the SIP.
After you create appropriate compensation controls, you need to verify gates, before
calculate compensation. Adjust the gate around the fluorescence-positive population
on the histogram (gate P2 on Fig. 3.2.11).
Fig. 3.2.11
Fluorescence-negative populations can be defined using the Unstained Control
Tube. If negative populations had not been already defined, you could define
them by creating an Interval Gate around the negative population for each
Stained Control Tube (Gate P3 on Fig. 3.2.11).
Generate the compensation matrix
After data has been recorded and gates have been adjusted, you are ready to calculate
compensation.
Go to Experiment > Compensation Setup > Calculate Compensation (Fig.3.2.12)
The software calculates the overlap as the median
fluorescence intensity (MFI) of the positive stained
control minus the MFI of the negative stained control
for each control in all channels. If there is a gated
unstained population in the Unstained Control tube
and a gated unstained population in the Stained
Control tube, the population in the Stained Control
tube will be used in the calculation.
Fig. 3.2.12
69
If the compensation calculation is successful, a dialog appears prompting you for the
name for the compensation setup. Enter a name, and click:
o
Link & Save—Links cytometer settings to the experiment and save the setup
to the compensation setup catalog.
o
Apply Only—Applies the cytometer settings to the experiment without saving
the settings to the compensation setup catalog.
o
Cancel—Dismisses the dialog without saving the setup.
In the link and save option you cannot change either the fluorescence PMT
voltages or the compensation values in a tube without data. You can only
adjust the scatter PMT voltages. After recording your sample the compensation
values are available for manual adjustments.
In the apply only option all the PMT voltages and compensation values can be
changed before recording the sample. However, do not forget that if you
change the fluorescence voltages, the compensation values are no longer
valid. You have to record again all the compensation controls with the same
voltages.
Record Samples
Select Tube_1
o
To see the dot plots created in the beginning you have to return to the Global
Worksheet, by clicking the icon highlighted in red on Fig. 3.2.11.
o
Or you can create a new specimen and record your samples.
Acquire and record your samples.
o
To view the compensation the option Enable Compensation should be ticked
(in the Compensation tab in the Cytometer Window) in all the sample tubes.
70
Exporting data from Diva as listmode (.fcs) files
When you have finished running all your controls and
samples, the data needs to be exported from the
software as flow cytometry standard, or listmode files
compatible with other flow software.
FCS3.0 files are compatible with newer analysis
software such as FlowJo.
FCS2.0 are an older file type, which cannot use 18-bit
data, and so has lower resolution. Fluorescence data
is exported in linear or log amplified depending how it
was collected but you cannot change from one to the
other post collection. Additionally, any compensation
added at the time of collection cannot be removed.
Select
the
experiment
from the Browser.
Right click--> Export-->
FCS files
Choose save path:
Save the Experiment in
D:\BDExport\FCS
You can hold down the Shift key and use left click to
select multiple experiments.
In the window that appears check that fcs 3.0 is
selected.
This only exports the raw data files; if you wish to
keep the gates/plots etc that you can re-import back
into Diva, you will need to export the whole
experiment as a .xml file. Please ask a member of
staff to help you with this.
All data is saved in the BDExport folder on the D:
drive of the computer. The FACS Lab staff create a
new folder each month where data will be exported
so the save path should look something like this:
D:\BDExport\FCS\2012\Aug 2012
Experiment completed.
You can analyse your results
in FlowJo or other analysis
programmes.
*You should not need to change anything; a new
folder with your name will be created automatically,
there is no need to type in your name and doing
so creates unnecessary folders.*
Diva will place all your experiments from one
month together in your folder (named as it
appears in the Browser window).
Data backup is currently performed by the FACSlab
and IS.
71
Working with templates
If you will be running the same experiment every time you use the LSR, or would like to
periodically run an experiment with the same antibodies, you may find it useful to export
the experiment as a template.
Creating a template allows you to save:
Plots used to look at samples (scatter, fluorescence etc).
Gates and gating strategies.
“Statistics View” display settings.
Fluorescence parameter labels (e.g. 530/30blue re-labelled to CD4-FITC will show up
as CD4-FITC 530/30blue on any plot axes).
Stopping gate settings (e.g. record 10K live cells).
Compensation previously applied using single colour controls.
Instrument settings such as PMT voltages.
For example, in the FACS Lab we commonly use DNA analysis templates from which we can
immediately open up an experiment displaying a scatter plot, PI-A vs PI-H (doublet
discrimination plot), PI-A DNA histogram, PI set to Linear, and stopping gate set to record
10K of scatter gated single events.
Once you have created an experiment using your template, you can tailor it specifically for
that day’s experiment. For example, the PMT voltages may not be quite right for the cells
you are running today (e.g. the scatter is slightly different because you are running a
different type of cell, or the staining has not worked quite as well so you need to increase a
fluorescence PMT voltage); bear in mind that compensation will need to be changed if you
change fluorescence PMT voltages. You can also add parameters to your experiment if you
have stained to look at an extra marker, and so forth.
*Important!*
Compensation should always be checked and not simply assumed to be correct!
Compensation can generally only be saved and re-used if you are using the same batch of
antibody (you should perform the experiment a few times including the single colour
controls to check that you get consistent results with your staining). If you get a new batch
of antibody, you will need to check with single colour controls that the compensation is still
accurate.
Changing any of the fluorescence PMT voltages will affect the compensation!
72
Creating a template
After you have set up and run the experiment for the first time, you should have an
experiment
(which
you
should
have
re-named
with
your
initials,
the
date,
and
LSRIIA/LSRIIB/Fortessa A/ Fortessa B) within YOUR FOLDER in the Browser (as described in
section 3.2).
*Saving a template*
Ensuring first that the experiment is open:
Right click on your experiment (book icon) in the Browser; select from the menu:
Export Experiment Template  Users
This opens a Wizard:
Rename your template; include your name or initials.
Change the template type to: “Users”. Click Finish.
The location of the template (e.g. should you want to delete it) on the D drive is:
D:\BDExport\Templates\ Experiment \ Users
* Using the template*
Click once on your folder to highlight the location to create an experiment.
Select the Experiment tab New Experiment (OR: right click your folder)
This opens a window showing all saved templates; choose yours from the list.
Remember to re-name the experiment within the Browser.
B
A
Fig. 3.2.9 Using a saved template to create a new experiment.
A Creating a new experiment in selected folder (in this case, the FACSlab folder).
B Selecting template: select Users tab and choose from list of saved templates.
73
Flow cytometers are complex instruments that require a precisely focused flow of cells
through a three dimensional space (the flow cell). Perfect function of fluidics is required
for the cells to correctly arrive at the laser interrogation point(s), and perfect alignment
of excitation and detection optics is required to capture the fluorescence emitted by cells
at these interrogation points with the greatest sensitivity, resolution and optimal signal
to noise ratio.
4.1 Routine Checks
In the FACS Lab, we routinely run checks to assess the performance of the cytometers.
Broadly these can be divided into alignment checks and sensitivity checks. Brightly
fluorescent beads, excited by each laser wavelength, are used to assure us that the
cytometer is aligned; i.e. that the laser is hitting the stream at the right place and the
fluorescence detection optics give the maximum signal. At a given voltage, the bead
peak will fall in a certain channel so we can plot this over days, weeks and months to
assess machine performance.
As well as alignment, we have to assess sensitivity; this is the cytometers ability to
resolve small differences in fluorescence, especially low levels of fluorescence. To do this
we use beads that are a mixture of levels of fluorescence (typically we use 8-peak beads
i.e. a solution that contains 8 different levels of fluorescence). Again, at given voltages
we know the best separation that can be achieved and this is monitored over time.
Fig. 4.1 8-peak beads used in quality control assurance.
74
Deterioration in either alignment or sensitivity will indicate that intervention is needed
and this is done either by members of the FACS Lab or by company field service
engineers.
4.2 Laser Delay, Area Scaling and Window
Extension
Fluorescent beads also help us to set laser delay, area scaling factor and window
extension. These are three parameters that are important but should not be altered
by users.
The lasers in the LSRs are spatially separated i.e. particles travel sequentially through
the beams. For example, in the LSRIIA the order is 488nm, 405nm, 355nm, 633nm. The
electronics need to be able to match signals to each laser; to do this, the distance
between each must be precisely known. We use fluorescent beads to check this. As
alignment in the LSR is fixed, this should not alter. However, if the flow cell becomes
dirty, particles take more time to pass between lasers and the timing therefore becomes
out of tolerance. Cleaning usually cures this. If you notice a problem, you should alert
any member of the FACS Lab.
The area scaling factor is used to ensure that area and height measurements are in the
same magnitude. The H signals generated by the electronics are fixed i.e. they will have
a value somewhere between 0 and 16,384 (214). A signals are the sum of all the
sampled height signals within the window gate and for a typical pulse is in the range of 0
to 262,144 (218), so a scaling factor has to be applied to the A signal. In practice, this
will make very little difference for most cell types and the value has been set to cover
the range seen in the Lab.
The window extension represents the amount of time during which a pulse from the
particle is sampled. Increasing the window extension extends the detection time to allow
for a more complete sampling of the signal pulse. However, if the window extension is
too large, more noise is included in the signal pulse, and the peak CV is increased.
75
Alternatively, if the window extension is too small, the pulse may not be completely
measured. On the LSR, this should not be adjusted by the user.
These parameters are routinely checked and adjusted if necessary by FACS Lab staff.
For more information please visit Practical Flow Cytometry” (Needs registration):
http://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-andservices/flow-cytometry/practical-flow-cytometry/index.htm
76
One of the basic requirements for flow cytometry is a single cell suspension free from
clumps. For cultured cell lines, there are two types: cells grown in suspension (e.g. B
and T cell lines) and adherent cells which may grow on feeder layers, coated surfaces or
straight onto plastic. Cells can also be obtained from whole blood or tissue samples.
5.1 Harvesting cells
Preparing
suspension
cells
basically
involves
taking
an
aliquot,
pelleting
by
centrifugation, washing and staining. Preparing cells from whole blood is similar to
working with cells in suspension, however a lysis step is usually required to remove red
blood cells as there are so many of them (problematic if you are trying to look at rarer
events if not lysed, and also very autofluorescent). They are also difficult to distinguish
from white blood cells using scatter.
Adherent cells should be harvested as for tissue culture. Typically this is with trypsin
(this is cell type dependent, fragile cells may also require gentler enzymes; there are
many commercially available cell dissociation solutions that you may wish to try). Cells
should be carefully pipetted to break up clumps, and filtered through a suitable sized
mesh (a cell strainer is the easiest way to do this). Take care also to not over digest as
this may cause damage and clumping.
*Surface Antigens*
If you are looking at surface antigens in adherent cells, be aware that some antigens
may be digested when harvesting cells for analysis, so you may have to test alternative
enzymes.
If the antigen is sensitive to all enzymatic/chelating treatments then you may have no
choice but to mechanically retrieve your cells by scraping with a rubber policeman or
cell scraper. This is quite a harsh method that often damages cells and produces clumps
that should be broken up by pipetting or passing through a 21 gauge needle followed by
filtering through mesh. Be aware, however, that you may still lose some of your cell
surface markers due to damage.
77
5.2 Resuspending cells for analysis
Suspension cells can be resuspended in PBS; (use Ca2+/Mg2+ free) these should not
clump unless there are a lot of dead cells (released DNA is sticky, treat with DNAse if
problematic). Adherent cells are often fine in PBS also, but sometimes a chelating agent,
EDTA, can be added (up to 5uM) to help inhibit the reforming of metal dependent
cell:cell interactions, if the cell type you are using has a tendency to clump. Foetal calf
serum may be added if viability is a problem but it must be kept to a minimum (less
than 2%), as excess of protein (which is sticky) will clog the machine and can also act as
a lens, affecting the scatter signals.
The way that you retrieve and resuspend your cells for analysis is really cell type
dependent, and it is always a good idea to talk to other members of your lab who may
have used the cell line you are using.
We can help with the retrieval of cells from various tissues/other 3-D structures (e.g.
embryoid bodies) or even paraffin sections. The FACS Lab staff are happy to offer
assistance with protocols to meet any sample preparation requirements you may have
and also to offer advice if you are having problems preparing your cells.
78
ADC: analogue to digital converter, electronic component that digitises an analogue
input signal (in this case the electrical signal received from the PMTs). Digitisation is
required for interfacing with the cytometer software.
analog: in cytometry, generally describes older electronics that measure signals as a
continuous spectrum, retaining information in the electronics while manipulations such
as gating are performed; ultimately the signal is digitised to interact with the software.
area measurement: integral of the area under the curve of an electrical pulse/signal,
describes the total fluorescence from one cell.
area scaling: use of a scaling factor which is applied to an electrical signal in order to
make use of full range of the 18-bit data scale available in the electronics.
autofluorescence: any fluorescence, generally due to cellular organelles and cyclic
structure molecules other than due to the fluorochrome of interest.
bandpass filter: optical filter which collects a narrow window of light.
baseline PMT voltage: minimum voltage that can be applied to a PMT before bottom end
sensitivity, and resolution is compromised.
benchtop: generally used to refer to analysers.
bi-exponential display (also logicle display): type of graphical display that has log scaling
at the top end, and linear around zero, negative events can be visualized instead of
piling up on the axis.
Clean (FACSClean): bleach cleaning agent manufactured by BD.
compensation: the mathematical subtraction (applied through the software by the
operator,
but
performed
by
hardware
components)
to
remove
a
particular
fluorochrome's emission that can be detected in channels other than its own.
comp
beads
(compensation
beads):a
type
of
antibody
capture
bead
made
of
polystyrene, manufactured by BD; available as Anti-Rat or Anti-mouse Ig, kappa. They
bind to rat or mouse light chain Ig and can be used instead of cells. Other commercially
produced capture beads are also available that can be used to aid compensation.
configuration: used to describe the optical system in a cytometer - the number of lasers
79
(and their wavelengths) number of detectors (and which optical filters) it is equipped
with.
contour probability plot: graphical display with contour levels drawn expressing
percentages of events falling at different levels, particularly useful for visualising the
distribution of events to determine how well-separated populations from closely drawn
gates will be.
core stream: stream of sample injected into the flow cell in the middle of the sheath;
undergoes hydrodynamic focussing.
CV: coefficient of variation; a measure of the dispersion of a data set. Commonly used in
flow cytometry to evaluate quality of DNA staining and to determine data file size
(number of target events required) in rare event analysis.
dead time: processing time during which a cytometer cannot handle information coming
from the next cell passing through the interrogation point(s).
detector: see also photomultiplier tube.
dichroic mirror: mirror which reflects specified wavelengths and may have long or short
pass function (reflecting light longer or shorter than specification).
digital: in flow, generally describes a cytometer with electronics that immediately
convert the analogue signal (from
the PMTs) into binary form
before further
manipulations are performed. This system is less vulnerable to noise, and cytometer
dead time is also reduced.
digitise: conversion of information to digital form - that is, binary code (0s and 1s).
Digital data has increased resolution if the bit size is increased (e.g. 10110011= 8 bit,
110101001110= 10 bit).
directly conjugated antibody: antibody directly linked to a fluorochrome, no secondary
antibody is required.
doublet discrimination: gating of single events for analysis, typically using pulse width
measurements.
event: particle of interest above threshold value passing through the primary laser and
80
triggering measurement
FACSFlow: BD brand sheath fluid for analysers; contains mild cleaning agents.
fcs file: flow cytometry standard, or “listmode” type of file.
flow cell: region in cytometer where hydrodynamic focusing occurs, a cuvette-type
structure in the LSRII.
forward scatter (see scatter): related to refractive index of the cytoplasm as well as cell
size.
Fluorescence Minus One (FMO or F-1): all colours MINUS one colour. Particularly
important for multi-colour flow; used to draw gates while taking into account nonspecific combinatory interactions of all fluorochromes used in an experiment. Each colour
used should have both a single colour control (for compensation) and an FMO (for
gating).
gate: a logical combination of regions.
height measurement: the height of the electrical pulse/signal, usually attributed to the
brightest spot of the particle.
histogram: single parameter graphical representation of data created by placing
numbers of cells (y-axis) in channels (x-axis) representing a range of (increasing)
values.
hydrodynamic focusing: fluid focusing by the addition of a sample into a faster flowing
sheath stream; used to constrict the core and promote a single celled stream that
passes through the interrogation points.
interrogation point: the laser intercept point, where fluorochromes from a particle of
interest become excited, emitting fluorescence which is then collected for analysis.
laminar flow: stable fluid flow in layers with no mixing.
laser delay: amount of time required a for particle to pass between each laser (due to
spatial separation of lasers) - allows hardware to determine which signal was generated
by which laser, on the basis of timing.
81
lookup table (LUT): data structure stored in computer memory – used in cytometers to
convert values to log.
longpass filter: transmits light longer than specified wavelength.
marker: equivalent of a gate, which can be drawn onto a histogram to define a region.
median fluorescence technique: a means to calculate correct compensation, by adding
compensation while observing median statistics for negative and positive populations in
channels that spill-over is being compensated out of, until the median values are equal.
noise: signals other than those coming from fluorescence light given off by particle of
interest.
octagon (PMTs): octagonal arrangement accomodating up to 8 detectors.
optical bench: the optical layout of the cytometer, a bench area comprising the lasers,
emission, collection and detection optics.
parameter: attribute that can be measured by the cytometer.
peak: pulse height measurement.
photomultiplier tube, PMT: device with a specially coated surface designed to collect
photons and from them generate an amplified electrical signal (electron flow).
population: 1) cells defined by a gated region, 2) cluster of cells apparent by eye, on a
plot.
precision: the accuracy of a cytometer, ability to make the same measurement on a
particle. consistently.
primary antibody: antibody directed against antigen of interest, requiring a secondary
fluorochrome linked antibody.
primary laser: laser used to trigger the cytometer electronics when a cell passes
through.
prime, priming: function on LSR that prepares the flow cell by emptying and slowly
refilling it, ensuring that it is free of air bubbles.
82
pulse width measurement: see also width measurement.
region: windows drawn by the user in the software to highlight cells of interest.
resolution: a cytometer’s ability to distinguish between slightly different levels of signal,
is improved by increased bit-size digitisation generated by better ADCs; closely tied in
with sensitivity and precision.
Rinse (FACSRinse): detergent cleaning agent manufactured by BD.
sample injection port (SIP): assembly on LSRII where sample tube is installed.
sample injection probe (also “sample probe”): metal tube on SIP which takes up sample
from facs tube.
scatter: light scattered by a particle, usually measured from the primary laser. May be
scattered forward (in the direction of the laser) or to the side (90° to direction of laser in
the same plane).
secondary antibody: fluorochrome linked antibody directed against species that primary
antibody was generated in, allows primary antibody to be detected and also has
amplifying effect.
sensitivity: a cytometer's ability to detect a dim positive signal over background/noise,
or the threshold set.
sheath: pressurised fluid that the sample is injected into; the sheath then carries the
sample to the flow cell for interrogation. In the FACS Lab, we use PBS as sheath fluid.
sheath line: tubing carrying sheath from the tank to the flow cell.
sheath filter: filter membrane that ensures PBS is free of particles.
shortpass filter: optical filter transmitting shorter than specified wavelength.
side scatter (see scatter): related to granularity or complexity of a particle, side scatter
increases in dead or dying cells; also changes when cells are fixed.
signal to noise ratio: ratio that compares a desirable signal (e.g. music) to undesirable
signals (such as background noise). The higher the value, the less obtrusive the
83
background noise is.
spectral overlap: emissions by a fluorochrome detected in ranges other than around its
peak. Emission curves are generally not Gaussian (bell-shaped), and usually trail off to
the lower energy end of the spectrum (often referred to as a "red-tail of emission") thus overlapping more from this side.
spill-over: also known as spectral-overlap.
tandem (tandem conjugate): pair of fluorochromes that have been chemically linked
together in such a way that they are spatially very close. This allows the "electron
donor", when excited, to transfer its excited state electrons to its partner (the recipient)
by resonance energy transfer. The recipient uses this energy to emit fluorescence in its
particular emission wavelength. This has been a means to create new fluorochromes by
combining ones that already exist. Tandem conjugates are sensitive to light and
oxidation and their quality should be monitored carefully.
trigon (PMTs): arrangement of three PMTs in collection optics.
threshold: channel number that needs to be exceeded in order for the electronics to
start measuring a pulse (i.e. to recognize that there is a cell passing the laser).
viability dye: dye that enters dying/dead cells which have compromised membranes,
allowing discrimination between live and dead populations.
width measurement: calculated value that is proportional to the amount of time a cell
takes to pass through a laser.
window extension: extension of the window of time in which a pulse is being sampled.
Too small a windows extension value may result in loss of information, but too large
means the electronics may still be sampling the first cell when the next cell arrives.
84
Symptom
Cause
Solution
Threshold setup
incorrectly.
No events displayed.
Sample injection
port clogged.
Pressure leak crack in FACS tube.
Run button not green.
Pressure leak –
instrument not
pressurised.
Threshold should be set around 10-15k on FSC.
Try these procedures (in order!!):
1-Pipette/filter – if sample has visible clumps.
2-Backflush (press Prime) the line.
If clog persists:
3-Run the cleaning protocol.
4.Apply a slight vacuum with a syringe.
If none of these steps work please ask a member of
FACS lab staff for help.
Transfer sample to a new tube.
Instrument not pressurised, check sheath tank lid
properly fastened and air line connected. Lift
pressure release valve to check if tank is
pressurising.
Crack in FACS tube, transfer sample to new tube.
Air in sheath filter - purge air (follow instructions
described previously).
DIVA not connecting to cytometer.
Interface
communication
failed.
Restart computer, if this doesn’t solve it, restart
BOTH instrument and computer.
If the problem persists ask a member of the FACS
lab staff for help.
Software glitch.
Re-export data and count files again.
Always check number of files!!
FCS files does not contain
uncompensated data.
Data exported as
FCS2 not FCS3.
Re-export in FCS3.
Check FCS2 not selected.
Loss of compensation in exported data
even if exported in FCS3.
Software glitch.
Try re-opening the experiment, re-export files.
Ask a member of staff if problem persists.
Unexpected loss of signal, unusual
compensation. (e.g. PerCP and APC)
Time delay issues
between lasers.
No fluorescence signal, or lower than
usual.
Incorrect filter in
front of PMT.
Not all .fcs files exported.
85
If problem occurs while using tandem conjugates,
check the dyes have not degraded; ask FACS staff.
Ask a member of the FACS lab staff for help.
Check the correct filter is in place and change if
necessary – ask a member of staff to demonstrate.