1. The document discusses best practices for flow cytometry analysis including understanding the optical bench and fluorochromes, setting proper compensation and negative controls, optimizing antibody labeling levels, and clearly designing gating strategies. 2. Key steps mentioned are knowing the instrument configuration, using appropriate isotype and internal negative controls, validating positive thresholds for each sample, and providing thorough information on plots and gating. 3. Maintaining clean acquisition, avoiding non-specific labeling, and selecting optimal conjugates are also emphasized for good flow cytometry analysis.

1. Analysis of Cell populations
By Flow cytometry
Claude LAMBERT
Labo Immunologie CHU Saint-Etienne
European Society of Clinical Cell analysis

3. Fluorochrome detection
1 parameter
Fluorochrome
Best conjugate
Compensations
Neg controls fmo
artifacts

4. 525 575 620 675 695 767
FITC
530/30
SignalSignal
Not all fluorescence is captured by your PMT

5. One fluorochrome
one spectrum
spectraviewer

6. Instrument
1 - Know your optical bench
NAVIOS Laser 405 nm, 40mW Laser 488nm, 22 mW Laser 638 nm, 25mW
FL9 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 R785
Filtres 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, 11mW
Horiz
V450
Horiz V500 FITC PE PerCP PE-Cy7 FL5 FL6
V450 V510 B530 B585 B670 B785 R660 R780filtres 450/50 510/50 f
i
530/30 585/42 670 LP 780/60 660/20 780/60
Band finale 425 - 475 502-535 B
a
515-545 564-606 670 - 735 750-810 650-670 750-810

7. Canto
Navios

8. Canto
Navios

9. Leaking
2 - Know your
fluorochromes

10. Compensations

11. 525 575 620 675 695 767
530+30
585+42
670 LP
780+60
FITC
PE
PE-TxR
PE-Cy5
PerCP
PerCP-Cy5.5
PE-Cy7
530/30
= 530/30 -

13. 525 575 620 675 695 767
530+30
585+42
670 LP
780+60
FITC
PE
PE-TxR
PE-Cy5
PerCP
PerCP-Cy5.5
PE-Cy7

15. Undercompensated

16. Compensations
Correct compensations
3 – Mind the compensations

17. Compensations
One representative conjugate
Strong labelling
Stable fluorescence
Possible surrogates
4 – Optimize your
compensation settings

18. Inter laser Compensations

19. Inter laser Compensations
exception
Blue laser
Red laser
5 – Mind inter
laser excitation

20. Optimum labelling

21. Fluorescence Intensité
Always be
saturated
Highest signal/noise ratio
Signal correlated to antigen density

23. SATURATION
6 - Consider the number of
cells you analyze
Cell number

24. 0.25 0.5 1 2 4 8 16 32 64 µl of
Antibody
Fluorescen
ce intensity
Saturation
Too much saturation
Rise non specific labelling
Optimum labelling intensity

25. 0.25 0.5 1 2 4 8 16 32 64 µl of
Antibody
Fluorescen
ce intensity
Find the best (minimal) saturation
TITRATION
7 – ALWAYS at SATURATION

26. 0.25 0.5 1 2 4 8 16 32 64 µl of
Antibody
Fluorescen
ce intensity
WASH
8 – Wishing reduce noise

27. Optimize your conjugate

28. JW Gratama Cytometry 1998
Antigen density
Assign copy numbers of PE molecules
bound per bead
Antiboy Bound per Cell

29. Bikoue A et al, Cytometry 1996; 26: 137-147
Rule 4 : marker densities
are not equivalents
9 – Mind marker
density on cells

30. Best conjugate
Fluorochroms are not equivalent

31. Best conjugate
10 - Stronger Fluorochroms
dimer staining
(lower density)

32. 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

33. Optimize your Dot plots

34. Dot plots are more informative
than histograms
3 populations
In fact
more than
3 populations
2 dim graphs are most precise Graphs

35. Other types of graphs
Level curves
better definiton of valley
12 – Take advantages of plot types

36. Sequential selection of population
Quantification of sub groups
gives % of cells
Yes …. But % of what??

37. Rule 6 :
Always make your
gating strategy
very clear

38. 28.3%
Gating Hierarchy

39. Graphs Conditionning
Mention
Parent
population (s)
28.3%
Proper
labelling info

40. IL-17
IL-22
% of what??

41. Kamnesh R. Pradhan, et col
Cytometry Part B 2011; 80B: 335–338
Gating strategy explained in a paper
13 – Clearly design & describe
gating strategy

42. Positivity

43. What is positive?
Decision threshold difficult
Reproducibility among different samples
even more difficult
Repeattibility between labs, operators..
14 – Be sure of positive threshold

44. NEGATIVITY
Cells alones
(auto-fluorescence)
But does not exclude non specific labelling??

45. non specific deposit on cells
PC5
For each label

46. Exclude non specific labelling
Isotype Controls =
Same provider
Same isotype
Same fluorochrome
Use non specific
Surrogate antibodies
The closest to the protocol
ECD
FITC
PE/RD1
PC5

47. Use cells in the sample
that you know they are negative for the
antibody considered
ECD
FITC
PE/RD1
PC5
PC5
Internal negative cell

48. Internal negative control
Use negative cells
(red) as negative
control..

49. 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

50. Follow acquisition
Exclude bubles
Temps

51. Surveiller acquisition
TAB
16 – Keep clean acquisition

52. 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 sampl
 Label saturation…. But not too much titration
 Avoid non specific labelling
 Gating strategy and graph information
 Optimize your conjugates

54. Design
Modern art