Orifice = 50 to 400 µ

Principles of Flow Cytometry

Quartznozzle

Fluorescence signalsFluorescence signals

Focalized laser beamFocalized laser beam

Injection of cells

Freq

Fluorescence

Photodiode

Light can be measured at 90° : Side scatter + Fluorescence

Side scatter reflects the cell content

LaserLaser

Fluorescence intensity

FITC

FIT

C

101 104103102

Relative fluorescence intensity

Nu

mb

er o

f E

ven

ts

FITC

FIT

CFITC

FITC

FITC

FITC

FIT

C

FITC

Basics of Flow CytometryBasics 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

FluidicsFluidics

OpticsOptics

ElectronicsElectronics

The automated Microscope

Waste

Detector& Counter

Sample

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!

1

Hydrodynamic focussing in the cuvette

SheathSample

SheathSample

Sample pressure low, small core stream. Good for DNA analysis

High sample pressure, broader core stream.Bad for DNA analysis

LOW HIGH

• 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

Summary

Basic opticsc

A system of prisms and lenses directs the laser light to the interrogation point in the cuvette

Laser delay

SheathSample

•Umožňuje cross beam kompenzaci

•Vyžaduje stabilní fluidics

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

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

How a voltage pulse from the PMT is generated

Voltage

LaserLaser

LaserLaser

LaserLaser

t

t

t

Voltage

Voltage

1.

2.

3.

Height, Area, and Width

Time (µs)

Volt

age

Pulse area(A)

Puls

e H

eig

ht

(H)

Pulse Width (W)

400

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.

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

An overview

YearInstrument

IntroducedMost Frequently

Heard Comments

1976 FACS II When can I get one?

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?

1980 FACS IV/440 Do you really need 4 colors?

2003 FACS Aria When can I get one?

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

What are the advantages / disadvantages?

• Advantages

• 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

Excitation- and Emission spectra of dyes for the blue laser

•Stejná excitace různá emise

•Překryv spekter(overlap)

•Excitace jiným laserem?

Compensation

www.bdbiosciences.com/spectra /

How much compensation is correct?

PE

PE

Importance of ACCURATE Compensation

RPCI

LFC

n = negativesd = dim positivesb = bright positives

10 1 10 2 10 3 10 4

FL3-Height -->

101

102

103

104

FL4

-->

PE-CY5-CD8

APC CD45

10 1 10 2 10 3 10 4

FL3-Height -->

101

102

103

104

FL4

-->

n d b n d b

10 1 10 2 10 3 10 4

FL3-Height -->

101

102

103

104

FL4

-->

Uncompensated Compensated Over Compensated

bn

Where is the CD8 dull population?!

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.

Hardware Compensation

How to set compensation on the instrument

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.

-Run unstained cells

-Adjust the PMT voltages so that the negative population is off the axis in every channel.

Setting compensation- PMT Voltage

FL1-no stain

FL

2-no

sta

in

Uncompensated Compensated

Median values both = ~3.2

Setting compensation - FITC single stain

FL1-FITC CD3

FL

2-no

sta

in

FL

2-no

sta

in

-Run single stained control (FITC stained only)

-Adjust the compensation value so that positive and negative population have the same FL2 median fluorescence intensity.

FL1-FITC CD3

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

Median values both = ~2.5

FL

2-P

E C

D4

FL1-no stain

Compensation Controls

Single Stain Controls

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

Single Stain Controls - Which cells?

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…

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

Single Stain Controls - which reagents?

Compensation of tandem-conjugates can differ from lot to

lot

• Use same reagent as experimental sample• Lots positive• Small CV, bright• Some reagents won’t work (IgL, EMA/PI)

– can mix with regular comps

Using Antibody Capture Beadsas single stained controls

Software Compensation

Automated Tools for Setting Compensation

Compensation Tools

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

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•Others

Compensation Matrix

FL1 FL2 FL3

FL1 Comp 3.96 0

FL2 Comp 27.35 5.15

FL3 Comp 0 11.18

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

Polychromatická cytometriePolychromatická cytometrie

Design experimentu a analýzaDesign experimentu a analýza

Childhood Leukemia Investigation Prague -Childhood Leukemia Investigation Prague -

Ústav imunologie, Ústav imunologie, Klinika dětské hematologie a onkologie, Klinika dětské hematologie a onkologie, UK 2.LF a FN MotolUK 2.LF a FN MotolPrahaPraha

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

Comparison of the dye intensity for the same marker

Baumgarth, Roederer, JIM, 2000, A practical approach to multicolor flow cytometry for immunophenotyping

Spektra fluorochromů

www.bdbiosciences.com/spectra /

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)

Zkreslení vlivem kompenzací

PE

FITC

PE

PE-TxRed

Přesvit (spilover, spectral overlap)z PE do FITC je malý = malá kompenzacez PE do PE-TxRed je velký = velká komp.

PE-TxRed – PE = 65%

Grafické řešení„Loglinear transformation“„Biexponencial display“

Zkreslení vlivem kompenzacíJe třeba promyslet odkud se dívat

Obvyklé problémy: PE vs PE-TxRed, PE-Cy5 vs APC

Nelze použít vždy histogram Nelze vždy použít čtverce či kvadrantyJe třeba promyslet jak postavit gate (kontroly FMO)V silně komp. kanálech je menší rozlišení a horší kvantifikace

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

PE PE-TxRed

•u CD4 PE pos. buněk CD8 PE-TxRed není

Jak naložit se zkreslením?

„donor“ „akceptor“

Na donor pozitivních buňkách se akceptor pozitivní znak nevyskytuje/nehodnotí

CD4 PE

CD

8 P

E-T

xRed

PE PE-TxRed

•znak s nízkou int. do PE

Jak naložit se zkreslením?

„donor“ „akceptor“

Nižší intenzita donoru = nižší rozptyl akceptoruCD3 PE

PE

-TxR

ed

PE PE-TxRed

vysoce exprimovaný znak do PE-TxRed

Jak naložit se zkreslením?

„donor“ „akceptor“

CD19 PE

CD

10 P

E-T

xRed

PE PE-TxRed

•kvalitativní znak do PE-TxRed

Jak naložit se zkreslením?

„donor“ „akceptor“

CD3 PE

CD

8 P

E-T

xRed

780/6

0735 LP

A

575/26

556 LP

D

488/10

-

F

H

695/

40

655

LP

B

610/20

595 LP

C

530/30

502 LP

E

G

PE-Cy7, PE-Alexa 750

PerCP-Cy5.5PerCP, PC5, Tricolor

Cy-Chrome PE-Dyomics647

PE-Texas RedECDPE –

Dyomics590

PE

FITC

Alexa 488

SSC

488nm Blue laser octagon

720/4

0680 LP

A

660/

20

-

B

C

Alexa 680

APC

Alexa 633

Alexa 647

Dyomics 647

633nm Red laser trigon

530/3

0502 LP

A

450/

40

-

B

C

Alexa Fluor 430

DAPI

Hoechst

Alexa 405

Pacific Blue

407nm Violet laser trigon