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Yan Deng (X4-5225), [email protected]
Gerald Denis (X4-1371), [email protected]
Mike Xie (X4-5225), [email protected]
for users of theFlow Cytometry Core Facility
at BUMC
21 September 2010
Introduction to the Principlesof Flow Cytometry
John Meyers (X8-7543), [email protected]
Flow Cytometry Core Facility: Personnel
Director of Cell Sorting Services: Yanhui Deng
Operations Manager: Mike Xie
Facility Consultant: John Meyers
Co-Director/Outreach: Gerald Denis
Director: David Sherr
http://www.bu.edu/cores/flow-cytometry
Where are the instruments located?
LSR II,MoFlo:670-5
FACScan:R901
FACScan:X620
FACScan,FACSCalibur:L50
8
http://www.bu.edu/cores/flow-cytometry
U R here
Flow cytometry was not easy in the old days
gendered division of labor
ungloved handsmissing PPE
live anthraxbacilli
But today… it’s as easy as PCR.
fluidics
optics
ele
ctro
nic
s3 elements of any
flow cytometry system
fluidics
FluidicsPurpose 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
sheath fluid
samplefluid
‘hydrodynamic focusing’
single file particles
● low flow rate
● narrow sample core
● high resolution
● high flow rate
● wide sample core
● low resolution
Always filter your samples to remove aggregates.
Fluidics
When conditions are right (i.e. when turbulence is minimal):
sample fluid flows in a central core
does not mix with the sheath fluid
This is termed ‘laminar flow’
optics
“SSC”
“FSC”
ele
ctro
nic
s
Forward and side scatter of leukemic cellsMalignant, large B cellsNormal B cells
FSC
SSC
The intensity of forward scatter light is proportional to size and cross-sectional area of the cells.The intensity of side scatter light is proportional tosize, shape and internal structure/irregularity of the cells.
Photomultiplier (PMT) detectors convert photons (selected by mirrors and filters) to electrical
pulsesPeak Height (volts)
Peak Width (time)
Peak Area
The higher the PMT voltage (user controllable), the greater the output magnitude for a given photon. At higher PMT voltages, the level of noise will also increase.
Adjusting the voltageof the PMT helps tooptimize captureof desired populations
Photomultiplier Tubes (PMTs)
FSC: forward scatter(size; cross-sectional area)
SSC: side scatter (granularity, internalor surface structurethat scatters light)
A dotplot represents two properties of a single cell
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SS
C-A
FSC
SSC
A histogram represents the distribution of a single parameter across many cells
0 102 103 104 105
APC-A: pH2AX
0
20
40
60
80
100
% o
f Ma
x
Control Condition Experimental10,000 cells each!
1 cell
1 cell
1 cell1 cell
Electronic processing of emission signals
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)
Gating
0 50K 100K 150K 200K 250KFSC-A
0
50K
100K
150K
200K
250K
SS
C-A
0 102 103 104 105
APC-A: pH2AX
0
50
100
150
# C
ells
Gating allows one to select populations based on computer or human-derived criteria and further gate or display the included
cells
Backgating – don’t lose your bearings!
Backgating allows one to determine if a gating strategy is all-inclusive of a desired cell type.
In the above example, some cells are missed! What are they?Many investigators overlook the importance of verification by backgating!
FLUORESCENCE
Excitation wavelength and emission wavelength areunique properties of each specific molecular structure
(FITC)
blue laser
Stokes Fluorescence
Excitation Emission
Stokes shift
Fluorescein (FITC)
Hoechst 33258 Texas Red
Propidium iodide (PI)
Laser light must overlap with excitation wavelength
yes
no
488
488
488
488
ex
ex ex
ex
em
em em
em
But different lasers are available to excite other molecules (LSR II)
488 nm (Blue) : FITC, GFP, PE, PerCP, PE-Cy5, PI, PerCP-Cy5.5, PE-Cy7
Widely-used molecules are excited by the 488 nm laser (FACScan)
355 nm (UV) : Indo-1, DAPI, Alexa Fluor 350, Hoechst 33258405 nm (Violet) : Alexa Fluor 430, Alexa Fluor 405, Pacific Blue 561 nm (Yellow/Green): Texas Red, Cherry Red, Tomato Red633 nm (Red): APC, APC-Cy7, Alexa Fluor 647, Alexa 680
bandpass filters
longpass dichroic mirrors
Octagon Detector Arrays
emitted fluorescent light
EMISSION
weakly expressedepitope
strongly expressedepitope
Fluorescence detection
Note logarithmic scale
autofluorescence
weakly expressedepitope
Isotype controls
Isotype control antibodies should be used at the same concentration to stain cells at the same cell density as the experimental, but they give fluorescent signals that define a negative result.
How do you knowit’s real?
isotype control test
Resolution sensitivity
Resolution sensitivity, the abilityto resolve a faint signal frombackground) depends on thedifference D between the positiveand background peaks and the spread of the backgroundpeak W
Reagent Stain Index
Phycoerythrin (PE) 356.3Alexa Fluor 647 313.1
APC 279.2PE-Cy7 278.5PE-Cy5 222.1
PerCP-Cy5.5 92.7PE-Alexa Fluor 610 80.4Alexa Fluor 488 75.4FITC 68.9PerCP 64.4APC-Cy7 42.2Alexa Fluor 700 39.9Pacific Blue 22.5AmCyan 20.2
Choose the right fluor for the job!
i.e. pick a bright fluor for a dim epitope
and avoid spillover of brightcell populations into detectorchannels that require highsensitivity for rare signals
Problems in Emission Fluorescence
Spectral overlap
Excitation Emission
Optical solutions to spectral overlap: Filters
Filters resolve overlapping wavelengths of emitted light
Longpass filter: transmits light of longer than or equal toa specific wavelength
Shortpass filter: transmits light of shorter than or equal toa specific wavelength
Bandpass filter: transmits light only within a narrow rangeof wavelengths
Examples of optical filters in flow cytometry
Optical detector configurations
octagon
660/20
APC
735 LP
780/60
APC-Cy7
red trigon
bandpass
bandpasslongpass
EMISSION
two bandpass filters
Electronic solutions to 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
Before After
Multicolor immunophenotyping
No antibody. Autofluorescence only. No compensation applied.
CD4-PE. No compensation applied.
CD4-PE. Correct compensation applied.
1.4% PE subtracted from FITC PMT, 6.5% PE subtracted from APC PMT.
CD8-FITC. No compensation applied.
CD8-FITC. Correct compensation applied.
12.5% FITC subtracted from PE PMT.
CD4-PE + CD8-FITC. Streptavidin-APC alone.
CD4-PE + CD8-FITC. CD3-biotin + Streptavidin-APC
3 COLORS, CORRECTLY COMPENSATED
Spectral overlap of some fluorochrome combinationscannot be compensated easily or at all
Cy5APC
Therefore, avoid such combinations
Contour plots provide more accurate data representation than dot plots
Gates
gra
nula
rity
→
size →
The uses of gates for cell sorting
FSC
SS
CC
D8
CD3
Four Applications
Multicolor immunophenotyping
Cell cycle analysis
Phosphoprotein and kinase signaling
Stem cell sorting by the “side population” method
Cell cycle analysis
7-aminoactinomycin D
an
ti-B
rdU
-FIT
C
(DN
A
syn
thesi
s)
DNA content (propidium iodide)
G0/G1
G2/M SG0/G1
G2/M
S
Linear detection
2N 2N 4N4N
Multicolor, Auto Compensation with Flow Jo
100 101 102 103 104
FITC
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
PE
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
Pacific Blue
0
20
40
60
80
100
% o
f M
ax
100 101 102 103 104
PE-Cy7
0
20
40
60
80
100
% o
f M
ax
isotype isotype
isotypeisotype
0 200 400 600 800 1000
100
101
102
103
104
gate 1:lymphocytes
gate 2:B cells
gates 3 – 5
up to 11 colors
gate 6:T cells?
etc
Filter configurations permit optimizationof multicolor stains
When two colors are not enough
A single PBMC sample simultaneously stained with antibodies to quantify expression of CD3, CD4, CD8, CD7, CD27, CD28, CD45RA, CD62Land CCR7.
A lymphocyte size gate and CD3+/CD4-/CD8+ color gate is appliedto characterize stages of T celldifferentiation.
The T cell compartment cannot be fully characterized by only 2 or 3 markers.
Current Protocols in Immunology 12:12 (2005)
Other markers
FluorochromeMarker
B220: B lineage
IgM: mature B cells
IgD: mature B cells
CD23: transitional B cells
AA4.1: transitional B cells
Pacific Blue
PE-Cy7
PE
FITC
APC
CD45.1: donor phenotype
CD11b/Mac-1: myeloid lineage
live/dead discrimination*
CD45.2-Biotin: recipient phenotype
CD3: T lineage
PerCP-Cy5.5
APC-Cy7
Invitrogen UV-activated vital dye
Streptavidin Alexa 350
Qdot 605
Example of a 10-color experiment on our LSR II
FITC
FITC
FITC Compensation Matrix
“Phosphoflow” techniques allow you to measure kinase cascades and signal transduction
CourtesyJohn Meyers
Hoechst red
Hoech
st
blu
e
0 64 128 192 256FL4
0
64
128
192
256
FL5
0 64 128 192 256FL4
0
64
128
192
256
FL5
0.25%
Control Verapamil
Identification of a Hoechst 33342-staining ‘side population’ from murine bone marrow
100% of the gated side population is also Sca1+ ; these are hematopoietic stem cells.
Kinetic identification of “side population” stem cells
These gated stem cells can be isolated by MoFlo
FCCF rates
http://www.bu.edu/cores/flow-cytometry
On-line schedulingActual cytometer and work station use tracked to the minute,
recorded in and billed monthly by enterprise class central server
EquipmentUnassisted
rate (per hour)Assisted rate
(per hour)MoFlo NA $80.00LSR II $45.00 $67.50
FACScans $40.00 $67.50FACScalibur $40.00 $67.50Workstations $10.00 NA
Help and training:
Please sign up for basic training on FACScan or LSR2
with Yan Deng (X4-5225), [email protected]
Problems during experiments:
We can’t read minds. Please write a computer entry
in the COMPLAINT LOG for each instrument. Or email us: [email protected]
We will contact you ASAP and usually can respond within 2 hours.
