Flow Cytometry
at
Boston University Medical Campus
Introduction to some methods that we offer
Yan Deng (X4-5225), [email protected]
Gerald Denis (X4-1371), [email protected]
Definitions
Flow cytometry simultaneously measures and analyzes
multiple physical characteristics of single particles,
usually cells, as they move in a fluid stream through a
beam of light.
Any suspended particle between 0.2 and 50μM is
suitable.
Larger particles, solid tissue or clumps of cells must be
disaggregated to be analyzed.
Examples: lymphocytes, protozoa, micron beads, chromosomes
Definitions
The particles in the fluid stream scatter incident light,
which reveals internal properties, size and granularity.
The particles also fluoresce;
they emit laser light at the interrogation point;
this light is picked up by detectors arrayed at a
different angle to detectors of scattered light.
fluidics
electronics
optics
Fluidics
waste
flow cell
laser
sheath fluid
sample
~2 x 105 to 1 x 107 cells/ml
Electronics
digitize
event
FSC
SSC
FL1
FL2
FL3
FL4
(size)
(granularity)
(green)
(yellow)
(red)
etc
1
500
300
638
840
20
-
2
200
100
245
85
50
-
3
600
800
300
700
30
-
sample
fluid
sheath
fluid
Fluidics
Purpose of the fluidics system:
1. Transport particles in a fluid stream
to the laser beam to be interrogated
2. Position the sample core in the center
of the laser beam
‘hydrodynamic focusing’
single file particles
● low flow rate
● high flow rate
● narrow sample
core
● wide sample
core
● high resolution
● low resolution
Fluidics
Concerns
1. Shear rates for cells: check after you complete
a run to ensure that the cells are intact.
2. Larger tips are needed for cell sorting.
Optics
The laser beam is focused on the sample core;
lasers must be fixed in place.
Excitation is accomplished by lasers that emit light at
specified wavelengths. The atomic properties of the
excitation media define this wavelength for a particular laser.
Argon blue lasers emit light at 488 nm.
This is the most common and versatile wavelength
for excitation of fluorochromes.
Laser light must overlap with excitation wavelength
Fluorescein (FITC)
Propidium iodide (PI)
Hoechst 33258
Texas Red
Optics
Filters resolve overlapping wavelengths of emitted light
Longpass filter: transmits light of longer than or equal to
a specific wavelength
Shortpass filter: transmits light of shorter than or equal to
a specific wavelength
Bandpass filter: transmits light only within a narrow range
of wavelengths
Excitation l of
Texas Red is not
optimal with 488 nm
laser
For more information
http://probes.invitrogen.com/resources/spectraviewer/
http://www.bdbiosciences.com/spectra/
Electronics
The electronic system:
1.
quantifies the voltage pulse
2.
converts analog signals to digital values
3.
performs compensation
4.
transfers data to the computer for analysis.
Electronics – Photomultiplier Tubes (PMTs)
Adjusting the voltage
of the PMT helps to
optimize capture
of desired populations
Electronics - Amplifiers
Amplifiers are of two types: linear or logarithmic
Linear amplification is typically used with scatter.
Logarithmic amplification is typically used with fluorescence.
DNA content
(Linear detection)
DNA content
(Log detection)
Electronics
Threshold is the minimum pulse height above which
a signal will be processed electronically.
Electronics
When a threshold value is defined, only signals with
intensities greater than or equal to the value are recorded.
before
after
Adjustment of blood immunophenotype to exclude debris
Fluorescence
Fluorescence
Fluorescence intensity is proportional to the number of binding sites
of the fluorescent compound, or “fluorochrome”.
Remember, when labeling live cells, to keep the experiment at 40C, because
receptors are internalized and recycled, leaving their fluorochromes behind.
Immunophenotyping
negative stain
positive stain
Cluster of Differentiation antibody specificity
CD3
CD4
CD5
CD11b
CD19
CD20
CD25
CD34
CD69
Pan-T lymphocytes
T helper/Inducer
T cells, subset B cells
Monocytes, granulocytes, NK/T cells
B cells
B cells
Il-2R, Tac IL-2 receptor, Activated T cells,
B cells, NK cells, monocytes
Progenitor cells
Early activation antigen on T, B and NK cells
Immunophenotyping
Two-color fluorescence of lymphocytes
1%
CD3-PE
44%
48%
7%
CD19-FITC
Note the experiment-specific definition of the “negative” population
Fluorescence
Multicolor analysis
Spectral overlap
Compensation
To correct for emission spillover of FITC signal (normally detected
in the FL1 channel) into the FL2 channel (which detects PE),
it is necessary to use filters or electronic compensation or both.
Uncompensated
Optimal
Compensation
Multicolor immunophenotyping
Before
After
Data presentation formats
Gates
Gates
The uses of gates for cell sorting
Some applications
Cell cycle kinetics
Multiparameter sorting
Some applications
DNA content for
cell cycle / apoptosis
Immune system activation