Shared Resources: Flow Cytometry and Cell Sorting
Standard Operating Procedure For:
Using the BD™ High Throughput Sampler (HTS):
BD LSRII (L01/L02)
Independent Training Manual Addendum
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Standard Operating Procedure
SOP Number: Independent End User Training
Curriculum and Supporting Materials
DHVI BD LSRII (L01/L02)
Effective Date: 8/8/2014
Addendum
Version Issue Number: 1
Owner:
Gregory D. Sempowski
Author(s):
Patrice McDermott
Melissa Ventevogel
Gregory D. Sempowski
Addendum:
Title: Using the BD™ High Throughput Sampler
(HTS): BD LSRII (L01/L02)
By signing the “Approved By” section below, the person attest that he/she has personally conducted a review of the
document for completeness and accuracy and approves the contents of the SOP document.
Approved By: Gregory D. Sempowski, PhD
Signed:
Title:
Flow Facility Director, DHVI
Scientific Director for Shared Resources, DHVI
Date:8/8/2014
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SOP Revision Document History:
Title &
Version
1.0
Replaces
NA
Effective
Date
8/8/14
8/29/14
Description of Change
New Procedure
Added 8.1.5.3
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Standard Operating Procedure For:
Using the BD™ High Throughput Sampler (HTS):
BD LSRII (L01/L02)
1.0
Purpose
The purpose of this procedure is to provide a comprehensive and consistent approach
to using the BD™ High Throughput Sampler (HTS; Plate Loader) on the DHVI Flow
Facility L01 and L02 bench-top analyzers.
2.0
Scope and Application
This SOP applies specifically to the BD LSRII (L01/L02) instruments in the
MSRBII Building, Hallway 2045, Bay 5 and 6. This procedure applies to Flow
Facility Independent End Users as well as Flow Facility Operator(s) assigned
responsibility for these cytometers. There is a baseline expectation that
Independent End Users and Operators are sufficiently trained and competent in
the operation of FACS Diva software and the instruments themselves. This
procedure details preparation for using the HTS, hooking up and running the
HTS, daily shutdown of the HTS and weekly shutdown of the HTS.
3.0
Reporting
This procedure applies to Flow Facility Independent End Users as well as
Operators who are assigned responsibility for this cytometer by the Facility
Director.
Report all problems with the HTS to a Flow Facility Operator, Dr. John
Whitesides (DHVI Director of Cytometry Innovation and Engineering or Dr. Greg
Sempowski (Flow Facility Director). See Facility Contact List on the wall between
Bay 5 and Bay 6 (and Attachment #2).
4.0
Safety
All activities performed under this SOP must be done in accordance with
applicable DHVI Safety SOPs. Current DHVI safety SOPs are on the N: Drive, in
Q Pulse, and are available on the DHVI Flow Facility website.
4.1
DHVISAFE_001*; DHVI SOP 001: General Biosafety Procedure
4.2
DHVIFLOW_013*; SOP Flow_013_Laser Safety
4.3
DHVIFLOW_014*; SOP Flow_014_General Safety
* Q pulse Document Name
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5.0
Introduction:
HTS allows quick conversion between tubes and plates. It provides fully
automated and rapid sample acquisition. It is designed to speed through a
microtiter plate in 15 minutes with sample carryover tolerances of 1% or less. It
supports standard 96-well U-, V-, and flat-bottom, and 384-well flat bottom plates.
6.0
Using the HTS unit:
6.1
Startup L01/L02 Instrument (if needed):
6.2
Setting up Diva Experiment for the HTS:
6.2.1 Set up the Experiment and run the control tubes for the experiment
as normally done.
6.2.1.1
NOTE: Manual setup is easier and faster when done in
tubes.
6.2.2 Click on the arrow next to the New Plate button in the Browser
toolbar and select a plate from the drop-down list.
6.2.3 NOTE: Make sure you choose the plate type that corresponds to
the plate you will be using. If you choose the wrong plate, the
probe could hit the plate between wells or strike the bottom of a
well, resulting in damage to the instrument and loss of sample.
6.2.4 Rename the Plate in the Browser just as you would a Specimen.
6.3
Installing the HTS on the LSRII:
6.3.1 Remove the back drip cup from under the Sample Injection Tube
(SIT; if applicable).
6.3.2 Remove the tube of water from the SIT and swing the aspirator arm
to the left.
6.3.3 Switch the acquisition control switch to Plate Mode.
6.3.4 Install the SIT protector by sliding the protector over the SIT, and
push up on the tube retainer until you can screw it onto the SIT and
tighten the tube retainer (if applicable).
6.3.5 Place the HTS on the base plate/carrier if not already there.
6.3.6 Remove the HTS safety cover.
6.3.7 Place the HTS on the LSRII.
6.3.8 Make sure the unit support legs fit into the bracket grooves. With
the legs in the grooves, slide the unit as far forward as possible.
6.3.9 Connect the Black power cable and the Silver communication cable
to their respective ports on the rear right side of the HTS unit.
6.3.10 Connect the sheath line (white) and waste line (orange) to their
respective ports on the LSRII cytometer.
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6.3.11 Connect the sample coupler to the SIT until you reach a hard stop.
6.3.12 Make sure the sample coupler tubing is not kinked or twisted. Hold
the coupler with one hand while you tighten the top nut with the
other hand.
6.3.13 Tighten the positioning screws on either side of the front of the
base plate/enclosure.
6.3.14 Slide the HTS unit all the way back toward the cytometer being
careful not to crimp the fluid tubing or communication cable.
6.3.15 Note: There should be a gap between the tightening nut and the
bottom of the SIT protector. If you do not see a gap, unscrew the
tube retainer, push the SIT protector all the way up, and retighten
the tube retainer (if applicable).
6.3.16 Turn on the HTS using the power switch that is located on the right
side of the HTS unit.
6.3.17 Put the cover on the HTS unit.
6.3.18 Put the LSRII in Low and Run mode.
6.3.19 In the Diva Workspace menu select > HTS > Prime.
6.3.20 After priming cytometer, the computer will show that the HTS is
ready.
7.0
Setting up the Plate in the Diva Software:
7.1.1 Highlight the Plate in the Browser.
7.1.2 In the Inspector frame, select Standard Throughput Mode.
7.1.3 Double click on the Plate in the Browser to open the Plate window
frame if not already open.
7.1.4 Click and drag to select the wells you want to acquire in and click
the Add Specimen Wells button to add a specimen to the plate
layout. Note: this is equivalent to creating a specimen with tubes
in it.
7.1.5 The Wells should turn blue with the Specimen Number in the upper
right hand corner. Wells will be acquired in the order they were
created/selected.
7.1.6 To change the name of the Specimen, click on the Specimen in the
List of Specimens on the Plate column and change the name or it
can be changed in the Inspector frame as well.
7.1.7 If you need to delete a Specimen, click on the Specimen in the List
of Specimens on the Plate table, right click > delete specimen.
7.1.8 If you need to delete a well, right click on the well > delete well(s).
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7.1.9 Label the wells just like you would your tubes using the Experiment
Layout window.
7.1.10 Select the Acquisition Tab in the Experiment Layout window and
select the number to record in each well.
8.0
Loader Settings for the Wells: The loader (HTS) settings shown in the Setup
view are the current settings for the selected well or plate. You can modify these
settings in either the Setup View column in the Plate window frame or in the
Inspector frame in the HTS tab.
8.1.1 Note: You cannot change HTS settings during acquisition or when
a sequence is in process.
8.1.2 Understanding Volumes: There are different volumes to take into
consideration when choosing loader settings. Figure 1-9 shows
different well volumes and Table 1-3 defines each volume type.
Volume Type Definitions::
Well volume: Volume that well can hold filled to the brim
Total volume: Volume pipetted into well – aspirated excess volume
Aspirated excess volume: Standard mode = 20uL High Throughput Mode = 22uL
Available volume: Volume pipetted into well – aspirated excess volume – dead volume
Minimum volume: 50uL for both standard and high-throughput modes for 96-well
plates.
Mixing volume: is one-half the available volume.
Dead volume: Volume in the bottom of the well that the probe cannot reach
8.1.3 Standard Throughput Mode: the HTS aspirates the selected
sample volume (2–200μL) plus an additional 2 μL from the well
and can process a 96-well plate in approximately 44 minutes. BD
recommends that you prepare your plate with a minimum of 250μL
of sample/well for a 96-well plate and 50μL/well for a 384-well plate.
8.1.4 High Throughput Mode: the HTS aspirates a fixed 22μL per well
even though you can select a sample volume between 2–10μL and
can process a 96-well plate in approximately 15 minutes. BD
recommends that you prepare your plate with a minimum of
100μL/well for a 96-well plate and 50μL/well for a 384-well plate.
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8.1.5 HTS Setting Definitions:
8.1.5.1
Sample Flow Rate is the speed the syringe aspirates
sample from the well in μL/second.
8.1.5.2
Sample Volume is the volume of sample aspirated from
each well for acquisition (Total volume pipetted into well
–minus the aspirated excess volume (20uL in Standard
mode and 22uL High throughput mode) – minus the
dead volume (30uL volume at bottom of well that the
probe cannot reach)).
Ex: 250μL-20uL-30μL = 200uL of sample volume
8.1.5.3
Stopping Time (secs) is determined by dividing the
Sample Volume by the Sample Flow Rate.
Ex: 200uL sample volume/2.0 Sample Flow Rate
=100 sec
Note: Data recording will stop when either the Events to
Record is met or the Stopping Time is met.
8.1.5.4
Mixing Volume is the volume of sample aspirated and
dispensed during mixing. The mixing volume should be
one half of the sample volume.
Ex: 250μL-20uL-30μL = 200μL/2 = 100μL
(Note: a mixing volume that is larger than the available
volume introduces air bubbles into the sample.)
8.1.5.5
Mixing Speed is the speed that the syringe aspirates
sample from and dispenses sample to the well during
mixing.
8.1.5.6
Number of Mixes is the number of mixing cycles that
are performed before a sample is aspirated.
8.1.5.7
Wash Volume is the volume of sheath fluid dispensed
for rinsing between wells to minimize carryover.
8.1.6 To achieve optimal throughput, BD recommends:
8.1.6.1
Concentrate the sample sufficiently to achieve your
number of events to record before the 200uL of sample
is finished.
8.1.6.2
Increase the Sample Rate to 1μL/sec or greater (Note:
the higher the sample flow rate, the lower the
resolution).
8.1.6.3
Set Sample Volume to a minimum of 250μL of
sample/well for a 96-well plate in standard mode.
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9.0
8.1.6.4
Set Mixing Volume to be one half of the available
volume (250μL-20uL-30μL = 200μL/2 = 100μL).
8.1.6.5
Use BD’s default Mixing Speed of 180 (Note: increased
mixing speed could compromise the separator bubble
between the sample and sheath, resulting in sporadic
event rates and possibly higher carryover).
8.1.6.6
Set Number of Mixes to 2 or less (Note: a greater
number of mixes could impact sample throughput).
8.1.6.7
Set Wash Volume to 400μL or less (Note: the greater
the wash volume, the slower the system throughput).
Acquiring Data:
9.1.1 During an uninterrupted sequence, BD FACSDiva software
calculates the acquisition time of each well based on sample
volume. This acquisition time is referred to as the stopping time,
and is calculated as follows: stop time (msec) = sample volume
(μL)/ flow rate (μL/sec) x 1 sec/1000 msec.
9.1.2 The software will stop acquisition or recording of a well and
proceed to the next well when any of the following stopping rules
have been met: the specified number of events is collected, the
stopping time is reached or the file exceeds memory specifications.
9.1.3 BD recommends you set the target number of events high enough
so that you do not run out of events before you reach the softwarebased acquisition stopping time.
9.1.4 Put your plate on the HTS plate holder with well A1 oriented to the
back right corner of the stage.
9.1.5 Put the HTS safety cover back on (Note: the HTS is equipped with
a safety interlock that prevents the cytometer from running when
the safety cover is removed or opened).
9.1.6 Select well A1 in the plate layout.
9.1.7 Put the LSRII in Low and Run mode.
9.1.8 Click Run Plate in the Acquisition Dashboard.
9.1.9 The sample probe goes through a homing sequence during
initialization.
9.1.10 The software automatically primes the HTS pumps during the
homing operation.
9.1.11 After the homing and priming operations, acquisition will begin.
9.1.12 Wells will be acquired in the order they were created.
9.1.13 At the end of the run, a dialog appears indicating the run is
complete.
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9.1.14 Click OK to dismiss the completion message.
9.1.15 Remove the safety cover and remove the plate.
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10.0
Pausing the HTS in the middle of a run:
10.1.1 To pause during a run, click Pause in the Acquisition Dashboard.
10.1.2 The HTS unit finishes processing the current well (or wells in high
throughput mode), and then remains in a suspended state until you
choose to continue.
10.1.3 To continue the run, click Resume.
10.1.4 To stop the run, click Stop Well(s) or Stop Plate.
10.1.5 This sequence allows you to stop the run early to minimize
additional sample loss.
11.0
Stopping the HTS in the middle of a run:
11.1.1 Click Stop Plate or Stop Well(s) in the Acquisition Dashboard.
11.1.2 The HTS finishes the sequence in progress, stops, and then the
following message appears: The Run Stopped.
11.1.3 Click OK.
11.1.4 Note: If you stop the HTS during a run in standard mode, the
current well will be lost. If you stop the HTS in high throughput
mode, the current and next well will be lost. Do not stop
acquisition if your sample volume is limited.
12.0
Daily Shutdown of the HTS: Needs to be done after every run.
12.1.1 The Cleaning plate is located on the shelf behind the L02 in Bay 6
above where the HTS is stored.
12.1.2 In the Diva Workspace menu > Select HTS > Daily Clean - 96-well
U bottom > OK.
12.1.3 Install the 96-well U bottom cleaning plate on the HTS with wells
A1-A4 filled with 10% Bleach and wells B1-B4 filled with DI water.
12.1.4 Click OK in the Confirm dialog box.
12.1.5 The cytometer goes through a homing sequence, and cleaning
begins. Note: the cleaning procedure can take up to 15 minutes.
12.1.6 Click OK when the HTS Clean Complete dialog box appears.
12.1.7 Remove the plate and rinse it out to be used next time.
12.1.8 Turn off the HTS using the power switch that is located on the right
side of the HTS unit and remove the HTS unit.
12.1.9 Switch the acquisition control switch to Tube mode.
12.1.10 Prime the cytometer a couple of times to remove any air bubbles
that may have gotten into the lines while running the HTS unit.
12.1.11 Put a tube of DI water on the SIP.
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12.1.12 Put the cytometer fluidics in Low and Standby mode.
13.0
Weekly Shutdown of the HTS:
Needs to be done after the cleaning procedure when the HTS will not be used
again that week.
13.1.1 Connect the white Accessory Tubing (located on the shelf behind
the L02 in Bay 6 above where the HTS is stored) to the Sheath
port on the cytometer.
13.1.2 Put the open end of the tubing into the bottle of DI water (located
on the shelf behind the L02 in Bay 6 above where the HTS is
stored).
13.1.3 In the Workspace menu > Select HTS > Prime.
13.1.4 Prime the system 3 times.
13.1.5
When finished, turn off the HTS using the power switch that is
located on the right side of the HTS unit and remove the HTS unit.
13.1.6 Switch the acquisition control switch to Tube mode.
13.1.7 Prime the cytometer a couple of times to remove any air bubbles
that may have gotten into the lines while running the HTS unit.
13.1.8 Put a tube of DI water on the SIP.
13.1.9 Put the cytometer fluidics in Low and Standby mode.
13.1.10 Shut down the cytometer (if applicable).
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Attachment #1: DHVI Flow Facility Contact List
DHVI Flow Contact List
General User Support:
FACMAN HELP:
MSRBII HELP
GHRB HELP
919-684-4130
919-684-3349
919-452-2709
919-452-1751
Labs/Instruments:
A01:
A02:
L01:
L02:
GHRB, 1071
MSRBII, 4036
MSRBII, Bay 5/6 Rm 2045
MSRBII, 4111
919-684-8813
919-613-5920
919-684-4130
919-684-4130
Role
FacMan Registration
Analyst (A01)
Analyst (CHAVI-ID)
Analyst (A02)
Scientific Advisor, CHAVI-ID
Billing/Finance
Facility Director
Director for Instrumentation
Faculty/Staff
Brantner
Cumming
Jones-Marshall
McDermott
Moody
Rusnak
Sempowski
Whitesides
Linda
Ian
Dawn
Patti
Tony
Joe
Greg
John
7/1/13
Office:
919-684-3349
919-684-9024
919-668-2636
919-684-4130
919-668-2551
919-681-2076
919-684-4386
919-684-4895
Cell:
919-451-8506
919-452-1751
919-949-4000
919-452-2709
919-332-9423
252-917-3477
919-699-4242
919-724-6451
Home:
Pager:
919-544-9884 919-970-9566
919-970-3685
919-381-5833
919-489-0891
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