1. Principles of Flow Cytometry
Injection of cells
Quartz
nozzle
Fluorescence signals
Focalized laser beam
Orifice = 50 to 400 µ

2. Light can be measured at 90° : Side scatter + Fluorescence
Photodiode
Laser
Side scatter reflects the cell content

3. Fluorescence intensity Relative fluorescence intensity
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Number of Events
FITC
FITC
101
102
103
104
Relative fluorescence intensity 6

4. Basics of Flow Cytometry
Cells in suspension
flow in single-file through
an illuminated volume where they
scatter light and emit fluorescence
that is collected, filtered and
converted to digital values
that are stored on a computer
Fluidics
Optics
Electronics

5. The automated Microscope
Detector
& Counter
This primitive diagram shows the principle: Cells are passing the microscope objective, and an electronic circuit decides whether the cells is fluorescent or not. This is how a flow cytometer works!
Waste
Sample

6. Hydrodynamic focussing in the cuvette
Sample
Sample
Sheath
Sheath
Sample pressure low, small core stream. Good for DNA analysis
High sample pressure, broader core stream.
Bad for DNA analysis
LOW
HIGH 1 1

7. Summary
Pressure (= Sheath Pressure) drives the sheath buffer through the cuvette, and the higher pressure in the sample tube (= Sample Differential) delivers the sample to the cuvette.
In the cuvette the principle of hydrodynamic focussing arranges the cells like pearls on a string before they arrive at the laser interception point for analysis
Hydrodynamic focussing cannot separate cell aggregates! Flow cytrometry is a technique that requires single cell suspensions 2

8. Basic opticsc
A system of prisms and lenses directs the laser light to the interrogation point in the cuvette

9. Laser delay Umožňuje cross beam kompenzaci Vyžaduje stabilní fluidics
Sheath
Sample
Umožňuje cross beam kompenzaci
Vyžaduje stabilní fluidics 1

10. Summary
Excitation light is steered with prisms and lenses to the interception point
Emitted light is collected using lenses and is split up with dichroic mirrors and filters 7

11. Tasks for the electronical system
Convert the optical signals into electonic signals (voltage pulses)
Digitise the data
Analyse Height (H), Width (W) and Area (A) of the pulse
Send the data to the analysis computer 8

12. How a voltage pulse from the PMT is generated
Laser t
Voltage 1. 2. 3.
Voltage 9

13. Height, Area, and Width Pulse area(A) Voltage Time (µs)
Pulse Height (H)
Voltage 40
Pulse Width (W)
Time (µs) 12

14. Threshold
The threshold defines the minimal signal intensity which has to be surpassed on a certain channel. All signals with a lower intensity are not displayed and not recorded for later analysis. 13

15. Summary
During passing the laser voltage pulses are generated at the PMT
Amplifiers enhance the signals
Only signals passing the desired threshold(s) are analysed and recorded
The data are finally passed to the analysis computer connected to the cytometer 15

16. An overview Year 1976 FACS II 1980 FACS IV/440 1991 FACS Vantage 1998
Instrument
Introduced
Most Frequently
Heard Comments
Year
1976
FACS II
When can I get one?
1980
FACS IV/440
Do you really need 4 colors?
1991
FACS Vantage
Do you really need 5 colors?
1998
FACS Vantage SE
Do you really need 6 colors?
2000
FACS DiVa
Do you really need 8 colors?
2003
FACS Aria
When can I get one? 1

17. Why always more colours?
More informations from Cell Phenotyping (Cell Surface Antigens)
around 300 CD Cell Surface Antigens
Many functional populations require 5 or more surface markers to be fully distinguished
Functional Assays
Cell Cycle (PI, BrdU, Intracellular Cyclins)
Apoptosis (Annexin-V, Active Caspase-3)
Ca++ Flux [Indo-1, FuraRed, Fluoro-4]
Cytokine Production
Intracellular Signaling (Rb phosphorylation)
Gene Reporter [Molecular] Assays
GFP, BFP, YFP, CFP Expression
LacZ Expression

21. What are the advantages / disadvantages?
Save Time and Samples
(1) 6-color stain = (15) 2-color stains
Exponential increase in information
Data from (1) 6-color stain » (15) 2-color stains
Identify new/rare populations (<0.05%)
Internal controls
Problems
Must carefully choose combinations of fluorochrome conjugates
Not all reagents are available in all colors
Greater potential for errors in compensation
Proper controls required

22. Excitation- and Emission spectra of dyes for the blue laser
Stejná excitace různá emise
Překryv spekter
(overlap)
Excitace jiným laserem?

23. Compensation /

24. How much compensation is correct?

25. Importance of ACCURATE Compensation
PE-CY5-CD8
APC CD45 n d b
Uncompensated
Compensated
Over Compensated
Where is the
CD8 dull population?!
n = negatives
d = dim positives
b = bright positives
RPCI
LFC

26. Which marker for compensation?
Small errors in compensation of a dim control (A)
can result in large compensation errors with bright reagents (B & C). Use bright markers to setup proper compensation.

27. Hardware Compensation
How to set compensation on the instrument

28. Setting compensation
Prepare single stained controls that have both a positive and negative population.
Adjust the PMT voltages so that the negative population is off the axis in every channel.
Align the centers of the positive and negative cell populations by matching the median fluorescence.

29. Setting compensation- PMT Voltage
-Run unstained cells
-Adjust the PMT voltages so that the negative
population is off the axis in every channel.
FL2-no stain
FL1-no stain

30. Setting compensation - FITC single stain
-Run single stained control (FITC stained only)
-Adjust the compensation value so that positive and negative population have the same FL2 median fluorescence intensity.
Uncompensated
Compensated
FL2-no stain
FL2-no stain
Median values
both = ~3.2
FL1-FITC CD3
FL1-FITC CD3

31. Setting compensation - PE single stain
-Run single stained control (PE stained only)
-Adjust the compensation value so that positive and negative population have the same FL1 median fluorescence intensity.
Compensated
FL2-PE CD4
Median values
both = ~2.5
FL1-no stain

32. Compensation Controls
Single Stain Controls

33. Single Stain Controls - Which cells?
Does not matter as long as:
The autofluorescence is the same in the negative and positive populations you are lining up.
eg, Pre-gate on lymphocytes if you are using CD8 FITC as a single stain control
The compensation values will be valid for ALL cell types, regardless of which type of cell is used to calculate the values.
The compensation is specific for the fluorochrome, not the cell type

34. Single Stain Controls - which reagents?
Use the same reagent (Ab-fluorochrome conjugate) as used in the experimental sample… OR
A different antibody may be substituted, as long as it is conjugated to the same fluorochrome.
However…

35. Single Stain Controls - which reagents?
Caveats for substituting reagents:
Controls should be as bright as possible
As bright or brighter than the experimental stains
GFP, CFSE, and FITC are NOT the same fluorochrome
even though they are all green!
With tandem dyes (Cy5PE/Cy7PE etc.) it is necessary to use the exact same reagent
spillover varies from reagent to reagent

36. Compensation of tandem-conjugates can differ from lot to lot

37. Using Antibody Capture Beads as single stained controls
Use same reagent as experimental sample
Lots positive
Small CV, bright
Some reagents won’t work (IgL, EMA/PI)
can mix with regular comps

38. Software Compensation
Automated Tools for Setting Compensation

39. Compensation Tools Must have single stained controls
Software calculated compensation for you!
Easy, accurate and quick.
Makes MULTI- Color compensation possible

40. Software Compensation Tools
Available on new generation machines
DakoCytomation’s Summit (version 4)
Coulter FC500
BD Diva
Others
Post-acquisition software
FCS Express
FCS Press
WinList
FlowJo

41. Compensation Matrix FL1 FL2 FL3 FL1 Comp 3.96 FL2 Comp 27.35 5.15
FL2 Comp
27.35
5.15
FL3 Comp
11.18

42. Compensation - Automatic Method
Automatic compensation in the Diva software offers a fast, easy and reliable method to set the correct compensation.
First, select „Create Compensation Tubes“ from the Instrument Menu:

43. Compensation - Automatic Method
The software automatically creates a list of single color tubes, based on your instrument setting.
Naturally, in certain experiments you may not want to use every channel for a multicolour experiment.
You can delete any tube from this list, but the corresponding channel is later, after automatic compensation, also deleted from your instrument setting!

44. Take Away Lessons Proper CONTROLS are essential
DON’T compensate by eye
Use Median to adjust the populations if you must do it manually
TRUST the software to do it for you
It does it quicker and more accurately

45. Polychromatická cytometrie
Design experimentu a analýza
Ústav imunologie,
Klinika dětské hematologie a onkologie,
UK 2.LF a FN Motol
Praha
Childhood Leukemia Investigation Prague -

46. Which fluorochrome to use?
Major Factors
Fluorochrome brightness
PerCP ≈ APC-Cy7 ≈ FITC << PerCP-Cy5.5 < PE-Cy7 < APC = PE-Cy5 < PE
Antigen density
Background staining of mAb
Inherent background (stickiness) of mAb
Antibody strength (Avidity)
Less antibody needed = less background
Amount of compensation required between conjugates
Single or multiple laser

47. Comparison of the dye intensity for the same marker
Baumgarth, Roederer, JIM, 2000, A practical approach to multicolor flow cytometry for immunophenotyping

48. Spektra fluorochromů /

49. Which fluorochrome for which marker?
In general, try to use brighter fluorochrome conjugates for duller antibodies or lower density antigens (e.g. activation antigens such as CD80, CD86, CD25, or CD28)
Use brighter reagents for staining cell populations with high autofluorescent backgrounds (e.g. granulocytes, monocytes, or activated lymphocytes)
Use duller conjugates (FITC or PerCP) for antigens expressed at high levels (e.g. B220 or CD4)

50. Zkreslení vlivem kompenzací
Přesvit (spilover, spectral overlap)
z PE do FITC je malý = malá kompenzace
z PE do PE-TxRed je velký = velká komp.
Grafické řešení
„Loglinear transformation“
„Biexponencial display“
FITC PE
PE-TxRed – PE = 65%
PE-TxRed PE

51. Zkreslení vlivem kompenzací
Je třeba promyslet odkud se dívat
Obvyklé problémy: PE vs PE-TxRed, PE-Cy5 vs APC
Nelze použít vždy histogram
Nelze vždy použít čtverce či kvadranty
Je třeba promyslet jak postavit gate (kontroly FMO)
V silně komp. kanálech je menší rozlišení a horší kvantifikace

52. Design experimentu Na interpretaci dat myslet PŘEDEM
Bez správných kontrol někdy interpretovat nelze
Jak naložit se zkreslením komp. dat?
Sestavit design experimentu tak,
aby se předešlo potížím při analýze

53. Jak naložit se zkreslením?
PE  PE-TxRed
„donor“
„akceptor“
Na donor pozitivních buňkách se akceptor pozitivní znak nevyskytuje/nehodnotí
u CD4 PE pos. buněk CD8 PE-TxRed není
CD8 PE-TxRed
CD4 PE

54. Jak naložit se zkreslením?
PE  PE-TxRed
„donor“
„akceptor“
CD3 PE
PE-TxRed
znak s nízkou int. do PE
Nižší intenzita donoru = nižší rozptyl akceptoru

55. Jak naložit se zkreslením?
PE  PE-TxRed
„donor“
„akceptor“
CD10 PE-TxRed
vysoce exprimovaný znak do PE-TxRed
CD19 PE

56. Jak naložit se zkreslením?
PE  PE-TxRed
„donor“
„akceptor“
CD8 PE-TxRed
kvalitativní znak do PE-TxRed
CD3 PE

57. 488nm Blue laser octagon PerCP-Cy5.5 PerCP, PC5, Tricolor Cy-Chrome PE
PE-Dyomics647 PE D
SSC B
575/26
695/40 F
556 LP
655 LP
488/10 - H G
502 LP
735 LP
780/60
530/30 A
595 LP
610/20
PE-Cy7, PE-Alexa 750 E
FITC
Alexa 488 C
PE-Texas Red
ECD
PE – Dyomics590

58. 633nm Red laser trigon APC Alexa 633 Alexa 647 Dyomics 647 Alexa 680 B
660/20 -
680 LP
720/40 A
Alexa 680 C

59. 407nm Violet laser trigon DAPI Hoechst Alexa 405 Pacific Blue
450/40 -
502 LP
530/30 A
Alexa Fluor 430 C