Analysis of Cell populationsBy Flow cytometry
Claude LAMBERTLabo Immunologie CHU Saint-Etienne
European Society of Clinical Cell analysis
Fluorochrome detection
1 parameter
FluorochromeBest conjugateCompensationsNeg controls fmoartifacts
525 575 620 675 695 767
FITC
530/30
SignalSignal
Not all fluorescence is captured by your PMT
One fluorochrome one spectrum
spectraviewer
Instrument
1 - Know your optical bench
NAVIOS Laser 405 nm, 40mW Laser 488nm, 22 mW Laser 638 nm, 25mWFL9 FL10 FITC PE PE-TEXAS RED PE-Cy5 PE-Cy7 APC ALEXA FL 500 APC-Cy7
V450 V550 B525 B575 B620 B675 B780 R660 R755 R785Filtres 450/40 550/40 525/40 575/30 620/30 695/30 755 LP 660/20 725/20 755 LP
Optical band 430 à 470 530 à 570 505 à 545 560 à 590 605 à 635 680 à 710 >755 650 à 670 715 à 735 755Mirors 480 SP 550 SP 595 SP 655 SP 730 SP 710 SP 750 SPBand finale 430 - 480 530-570 505 à 545 560-590 605-635 680-710 >755 650-710 715 à 735 755
Canto II Laser 405 nm, 40mW Laser 488nm, 13 mW Laser 633 nm, 11mWHoriz V450
Horiz V500 FITC PE PerCP PE-Cy7 FL5 FL6
V450 V510 B530 B585 B670 B785 R660 R780filtres 450/50 510/50 fi
530/30 585/42 670 LP 780/60 660/20 780/60
Band finale 425 - 475 502-535 Ba
515-545 564-606 670 - 735 750-810 650-670 750-810
Canto
Navios
Canto
Navios
Leaking
2 - Know your fluorochromes
Compensations
525 575 620 675 695 767
530+30585+42
670 LP780+60
FITC
PE
PE-TxR
PE-Cy5
PerCP
PerCP-Cy5.5
PE-Cy7
530/30
= 530/30 -
525 575 620 675 695 767
530+30585+42
670 LP780+60
FITC
PE
PE-TxR
PE-Cy5
PerCP
PerCP-Cy5.5
PE-Cy7
Undercompensated
Compensations
Correct compensations
3 – Mind the compensations
Compensations
One representative conjugateStrong labellingStable fluorescencePossible surrogates
4 – Optimize your compensation settings
Inter laser Compensations
Inter laser Compensationsexception
Blue laser
Red laser
5 – Mind inter laser excitation
Optimum labelling
Fluorescence Intensité
Always be saturated
Highest signal/noise ratioSignal correlated to antigen density
SATURATION
6 - Consider the number of cells you analyze
Cell number
0.25 0.5 1 2 4 8 16 32 64 µl of Antibody
Fluorescence intensity
Saturation
Too much saturation Rise non specific labelling
Optimum labelling intensity
0.25 0.5 1 2 4 8 16 32 64 µl of Antibody
Fluorescence intensity
Find the best (minimal) saturationTITRATION
7 – ALWAYS at SATURATION
0.25 0.5 1 2 4 8 16 32 64 µl of Antibody
Fluorescence intensity
WASH
8 – Wishing reduce noise
Optimize your conjugate
JW Gratama Cytometry 1998
Antigen densityAssign copy numbers of PE molecules bound per bead
Antiboy Bound per Cell
Bikoue A et al, Cytometry 1996; 26: 137-147
Rule 4 : marker densities are not equivalents
9 – Mind marker density on cells
Best conjugate
Fluorochroms are not equivalent
Best conjugate
10 - Stronger Fluorochroms dimer staining (lower density)
Visualization of Spectral Overlap
Gallios/Navios Flow Cytometers
2 types of distortion:
‚Silent‘ Fluorochromes:• bright markers (gating)
‚Untouchable‘ Channels• dimly expressed antigens
depends on instrument configuration, and fluorochromes.
11 – dim expression : stronger and untouchable fluorochome
Optimize your Dot plots
Dot plots are more informative than histograms
3 populations
In fact more than 3 populations
2 dim graphs are most precise Graphs
Other types of graphs
Level curvesbetter definiton of valley
12 – Take advantages of plot types
Sequential selection of population
Quantification of sub groups
gives % of cells
Yes …. But % of what??
Rule 6 : Always make your gating strategy very clear
28.3%
Gating Hierarchy
Graphs ConditionningMention
Parent population (s)
28.3%
Proper labelling info
IL-17
IL-2
2
% of what??
Kamnesh R. Pradhan, et col Cytometry Part B 2011; 80B: 335–338
Gating strategy explained in a paper
13 – Clearly design & describe gating strategy
Positivity
What is positive?
Decision threshold difficult
Reproducibility among different samples even more difficult
Repeattibility between labs, operators..
14 – Be sure of positive threshold
NEGATIVITY
Cells alones(auto-fluorescence)
But does not exclude non specific labelling??
non specific deposit on cells
PC5
For each label
Exclude non specific labelling
Isotype Controls =Same providerSame isotype
Same fluorochrome
Use non specific Surrogate antibodiesThe closest to the protocol
ECD
FITC
PE/RD1PC5
Use cells in the sample that you know they are negative for the
antibody considered
ECD
FITC
PE/RD1PC5
PC5
Internal negative cell
Internal negative control
Use negative cells (red) as negative control..
Validate negativity Inside the multiple labelling
FULL FLUORESCENCE MINUS ONE (FMO)Test one color negativity while all other labelings are there
All except CD19 All except CD8
15 – Be sure on the negative
Follow acquisition
Exclude bublesTemps
Surveiller acquisition
TAB
16 – Keep clean acquisition
Good Flowcytometry analysis Knowing your Optical scheme
Good Negative controls (including FMO)
Proper settings and do not change it
Positive threshold to be validated on each sample
Label saturation…. But not too much titration
Avoid non specific labelling
Gating strategy and graph information
Optimize your conjugates
Design
Modern art