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Quartz nozzle. Fluorescence signals. Focalized laser beam. Principles of Flow Cytometry. Injection of cells. Orifice = 50 to 400 µ. Photodiode. Fluorescence. Light can be measured at 90° : Side scatter + Fluorescence. Laser. Side scatter reflects the cell content. FITC. FITC. FITC.

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principles of flow cytometry

Quartz

nozzle

Fluorescence signals

Focalized laser beam

Principles of Flow Cytometry

Injection of cells

Orifice = 50 to 400 µ

slide2

Photodiode

Fluorescence

Light can be measured at 90° :

Side scatter + Fluorescence

Laser

Side scatter reflects the cell content

slide3

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

FITC

Fluorescence intensity

Number of Events

101

102

103

104

Relative fluorescence intensity

basics of flow cytometry
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

the automated microscope
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

hydrodynamic focussing in the cuvette

LOW

HIGH

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

1

slide7

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
slide8

Basic opticsc

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

laser delay

Sample

Sheath

Laser delay
  • Umožňuje cross beam kompenzaci
  • Vyžaduje stabilní fluidics
summary
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
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

t

1.

2.

3.

Laser

Voltage

t

Laser

Voltage

t

Laser

How a voltage pulse from the PMT is generated

Voltage

height area and width
Height, Area, and Width

Pulse area(A)

Pulse Height (H)

Voltage

0

40

Time (µs)

Pulse Width (W)

threshold
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.

summary15
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

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?

2003

2000

FACS Aria

FACS DiVa

When can I get one?

Do you really need 8 colors?

An overview

Instrument

Introduced

Most Frequently

Heard Comments

Year

why always more colours
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
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
Excitation- and Emissionspectra of dyes for the blue laser
  • Stejná excitace různá emise
  • Překryv spekter
  • (overlap)
  • Excitace jiným laserem?
compensation
Compensation

www.bdbiosciences.com/spectra /

slide25

Uncompensated

Compensated

Over Compensated

n

d

b

n

d

b

n

b

APC CD45

Where is the

CD8 dull population?!

PE-CY5-CD8

RPCI

LFC

Importance of ACCURATE Compensation

n = negatives

d = dim positives

b = bright positives

which marker for compensation
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

Hardware Compensation

How to set compensation on the instrument

setting compensation
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.
setting compensation pmt voltage
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

setting compensation fitc single stain
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

setting compensation pe single stain
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

compensation controls

Compensation Controls

Single Stain Controls

slide33

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
slide34

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…

single stain controls which reagents
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
slide37

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
software compensation

Software Compensation

Automated Tools for Setting Compensation

compensation tools
Compensation Tools
  • Must have single stained controls
  • Software calculated compensation for you!
  • Easy, accurate and quick.
  • Makes MULTI- Color compensation possible
software compensation tools
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
take away lessons
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
polychromatick cytometrie
Polychromatická cytometrie

Design experimentu a analýza

Ústav imunologie,

Klinika dětské hematologie a onkologie,

UK 2.LF a FN Motol

Praha

Childhood Leukemia Investigation Prague -

which fluorochrome to use
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
Comparison of the dye intensity for the same marker

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

spektra fluorochrom
Spektra fluorochromů

www.bdbiosciences.com/spectra /

which fluorochrome for which marker
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)
zkreslen vlivem kompenzac

Grafické řešení

„Loglinear transformation“

„Biexponencial display“

PE-TxRed – PE = 65%

Zkreslení vlivem kompenzací

Přesvit (spilover, spectral overlap)

z PE do FITC je malý = malá kompenzace

z PE do PE-TxRed je velký = velká komp.

FITC

PE

PE-TxRed

PE

zkreslen vlivem kompenzac49
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

design experimentu
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

jak nalo it se zkreslen m
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

jak nalo it se zkreslen m52
Jak naložit se zkreslením?

PE-TxRed

CD3 PE

PE  PE-TxRed

„donor“

„akceptor“

  • znak s nízkou int. do PE

Nižší intenzita donoru = nižší rozptyl akceptoru

jak nalo it se zkreslen m53
Jak naložit se zkreslením?

PE  PE-TxRed

„donor“

„akceptor“

CD10 PE-TxRed

vysoce exprimovaný znak do PE-TxRed

CD19 PE

jak nalo it se zkreslen m54
Jak naložit se zkreslením?

PE  PE-TxRed

„donor“

„akceptor“

CD8 PE-TxRed

  • kvalitativní znak do PE-TxRed

CD3 PE

slide55

488nm Blue laser octagon

PerCP-Cy5.5

PerCP, PC5, Tricolor

Cy-Chrome

PE-Dyomics647

PE

D

SSC

B

575/26

F

695/40

556 LP

655 LP

488/10

-

H

G

502 LP

735 LP

780/60

A

530/30

595 LP

610/20

PE-Cy7, PE-Alexa 750

E

FITC

Alexa 488

C

PE-Texas Red

ECD

PE – Dyomics590

slide56

633nm Red laser trigon

APC

Alexa 633

Alexa 647

Dyomics 647

B

660/20

-

680 LP

720/40

A

Alexa 680

C

slide57

407nm Violet laser trigon

DAPI

Hoechst

Alexa 405

Pacific Blue

B

450/40

-

502 LP

530/30

A

Alexa Fluor 430

C

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