Light scatterLight scatter

Forward angle light scatterForward angle light scatter in a narrow angle from the direction of the laser in a narrow angle from the direction of the laser

beambeam FALS or FSFALS or FS

Right angle light scatterRight angle light scatter at right angles to the laser beamat right angles to the laser beam RALS or SS (side scatter)RALS or SS (side scatter)

laserlaser

Forward light scatterForward light scatter

FS FS detectordetector

blocker barblocker bar

Light ScatterLight Scatter

The intensity of scatter is proportional to The intensity of scatter is proportional to the size, shape and optical homogeneity of the size, shape and optical homogeneity of cells (or other particles)cells (or other particles)

It is strongly dependent on the angle over It is strongly dependent on the angle over which it is measuredwhich it is measured particularly with forward scatterparticularly with forward scatter

Light ScatterLight Scatter

Forward scatter tends to be more sensitive to Forward scatter tends to be more sensitive to the size and the size and surface properties surface properties can be used to distinguishcan be used to distinguish live live fromfrom dead dead cells cells

Side scatter tends to be more sensitive to Side scatter tends to be more sensitive to inclusions within inclusions within cellscells can be used to distinguish can be used to distinguish granulatedgranulated cells from cells from

non-granulatednon-granulated cells cells

GatingGating

Set a region on a histogram or cytogramSet a region on a histogram or cytogram IF cell IN region THEN show another IF cell IN region THEN show another

propertyproperty

Cell selection by gatingCell selection by gating‘‘Gating’ on the lymphocytes. IF cell has light Gating’ on the lymphocytes. IF cell has light scatter in R1 THEN show on CD4/CD8 scatter in R1 THEN show on CD4/CD8 cytogramcytogram

lymphocytes

Triggering the electronicsTriggering the electronics

sign

al

time

threshold

Changing the threshold settingChanging the threshold setting

debris

I

distancecross-section

spherical

elliptical

Shape of the laser beamShape of the laser beam

Focus the laser beam with:Focus the laser beam with: spherical lens - circular cross-sectionspherical lens - circular cross-section cross cylindrical lens pair - elliptical X-sectioncross cylindrical lens pair - elliptical X-section

Pulse shape analysisPulse shape analysis

signal

time

laserlaser

flow

Integrated area =Integrated area =total fluorescencetotal fluorescence

Signal Signal peakpeakSignal width =Signal width =beam width + cell diam.beam width + cell diam.

Pulse shape analysisPulse shape analysis

single cells

two cells

DNA analysis by flow DNA analysis by flow cytometrycytometry

Michael G. OrmerodMichael G. Ormerod

m.g.ormerod@btinternet.comm.g.ormerod@btinternet.com

DNA contentDNA content

PloidyPloidy

Cell cycleCell cycle

DNA ProbesDNA Probes

Use DNA probes that are sUse DNA probes that are stoichiometrictoichiometric - - that is, the number of molecules of that is, the number of molecules of probe bound is equivalent to the probe bound is equivalent to the quantity of DNAquantity of DNA

Dyes for DNA cell cycle Dyes for DNA cell cycle analysisanalysis

Propidium iodidePropidium iodide Excited at 488 nm; fluoresces red (617 nm)Excited at 488 nm; fluoresces red (617 nm) easily combined with fluorescein staineasily combined with fluorescein stain also stains RNAalso stains RNA

DRAQ5DRAQ5 Max. excitation at 646 nm; can be excited at Max. excitation at 646 nm; can be excited at

488 nm; fluoresces in deep red at 680 nm max488 nm; fluoresces in deep red at 680 nm max Taken up by live cellsTaken up by live cells

Dyes for DNA cell cycle analysisDyes for DNA cell cycle analysis

Hoechst dyesHoechst dyes excited by uv; fluoresce blueexcited by uv; fluoresce blue DNA specific - bind to ATDNA specific - bind to AT Hoechst 33342 can be used to stain viable cellsHoechst 33342 can be used to stain viable cells

DAPIDAPI excited by uv; fluoresce blueexcited by uv; fluoresce blue DNA specificDNA specific

Definitions & TermsDefinitions & Terms DNA PloidyDNA Ploidy

Related to the quantity of DNA in a cellRelated to the quantity of DNA in a cell

DNA IndexDNA Index Ratio between the mean DNA content of the Ratio between the mean DNA content of the

test cells to the mean DNA content of normal test cells to the mean DNA content of normal diploid cells, in G0/G1phasediploid cells, in G0/G1phase

Coefficient of Variation (CV)Coefficient of Variation (CV) 100*SD/mean DNA100*SD/mean DNA Usually measured on G1/G0 cellsUsually measured on G1/G0 cells

FNA of human breast carcinoma PI stainFNA of human breast carcinoma PI stain

DNA

Cel

l num

ber

normaltumour

G1

G2S

DNA content - measuring ploidy & DNA content - measuring ploidy & SPFSPF

DNA analysis of the cell cycleDNA analysis of the cell cycle

Following changes in the cell cycleFollowing changes in the cell cycle

Quality control of DNA Quality control of DNA measurementmeasurement

Sample preparationSample preparation Instrument alignmentInstrument alignment Correct data analysisCorrect data analysis

Using propidium iodide for Using propidium iodide for DNA analysisDNA analysis

Excited at 488 nm (argon-ion)Excited at 488 nm (argon-ion) Fluoresces red Fluoresces red Does not cross intact plasma membraneDoes not cross intact plasma membrane

Permeabilise with detergent orPermeabilise with detergent or Fix in 70% ethanol orFix in 70% ethanol or Fix in paraformaldehyde followed by ethanolFix in paraformaldehyde followed by ethanol

Treat with RNaseTreat with RNase

Sample preparation for DNA analysisSample preparation for DNA analysis

Fixed cellsFixed cells Samples can be storedSamples can be stored Needed when adding antibody stainNeeded when adding antibody stain Quality may be reducedQuality may be reduced

Permeabilisied cells or nucleiPermeabilisied cells or nuclei Use fresh or frozen samples, limited storage Use fresh or frozen samples, limited storage

timetime High quality achievable (Vindelov method)High quality achievable (Vindelov method)

DNA measurementDNA measurement

Use linear amplificationUse linear amplification Cell cycle is linear, not logarithmicCell cycle is linear, not logarithmic Relevant information occupies more of the Relevant information occupies more of the

histogramhistogram Cell cycle algorithms assume a linear scaleCell cycle algorithms assume a linear scale

Instrument alignmentInstrument alignment

Check daily using standard fluorescent Check daily using standard fluorescent beadsbeads

Correct alignment essentialCorrect alignment essential (Some misalignment can be tolerated with (Some misalignment can be tolerated with

immunofluoresence measurement - not immunofluoresence measurement - not DNA)DNA)

DNA measurementDNA measurement

Use linear amplificationUse linear amplification Cell cycle is linear, not logarithmicCell cycle is linear, not logarithmic Relevant information occupies more of the Relevant information occupies more of the

histogramhistogram Cell cycle algorithms assume a linear scaleCell cycle algorithms assume a linear scale

Quality control of DNA measurementQuality control of DNA measurement

Measure the spread of the distribution across Measure the spread of the distribution across the G1/G0 peak as coefficient of variation the G1/G0 peak as coefficient of variation (cv)(cv)

C T

D C, T cv = 1%

D cv = 1.2%

DNA measurementDNA measurementHuman breast carcinoma cells prepared by the Vindelov Human breast carcinoma cells prepared by the Vindelov

method. PI stain.method. PI stain. (Data supplied by Gyda Otteson & Ib Christensen, Finsen Laboratory, Copenhagen)(Data supplied by Gyda Otteson & Ib Christensen, Finsen Laboratory, Copenhagen)

ATC

D

C 1.2%T 1.0%D 1.0%A 2.2%

DNA histogramDNA histogram

Measure DNA content Measure DNA content Problem with clumpsProblem with clumps 2 cells in G1 = 1 cell in G22 cells in G1 = 1 cell in G2 Distinguish by pulse shape analysisDistinguish by pulse shape analysis

Shape of the laser beamShape of the laser beamfocus with:focus with:

spherical lens - circular cross-sectionspherical lens - circular cross-section cross cylindrical lens pair - elliptical cross-cross cylindrical lens pair - elliptical cross-

sectionsection

I

distancecross-section

spherical

elliptical

Flow CytometryFlow Cytometry Pulse shape analysisPulse shape analysis

Integrated area =total fluorescence

Signal peakSignal width =beam width + cell diam.

PMTvoltage

time

cell

beam

Pulse shape analysisPulse shape analysis

signal

time

G1G1 G2G2 2 x G12 x G1

laserlaser

flow

2x

areaareahtht

widthwidth

DN

A p

eak

DNA area

clumps

singl

e

Pulse shape analysisPulse shape analysis

DN

A w

idth

DNA area

clumpssingle

ungatedungated

gatedgated

Pulse shape analysisPulse shape analysis

Measuring cell proliferation Measuring cell proliferation using the BrdUrd/anti-BrdUrd using the BrdUrd/anti-BrdUrd

methodmethod

Measuring cell proliferationMeasuring cell proliferation

DNA histogramDNA histogram BrdUrd/anti-BrdUrdBrdUrd/anti-BrdUrd Hoechst/PI/BrdUrdHoechst/PI/BrdUrd Dilution of labelDilution of label

DNA histogramDNA histogram

Static measurement of the cell cycleStatic measurement of the cell cycle First choiceFirst choice Easy to combine with antibody stainEasy to combine with antibody stain

CisplatinCisplatin

Following changes in the cell cycleFollowing changes in the cell cycle

Genotoxic Genotoxic drugdrug

S phase slow down

G2 block

BrdUrd/anti-BrdUrdBrdUrd/anti-BrdUrd

Pulse label with BrdUrd (30 min)Pulse label with BrdUrd (30 min) Harvest cells at different timesHarvest cells at different times Fix cellsFix cells Denature DNA (acid, heat or UV)Denature DNA (acid, heat or UV) Label with anti-BrdUrd and PILabel with anti-BrdUrd and PI

V79 cells(data supplied by G. D. Wilson,, CRC Gray Laboratories)

Brd

Urd

/FIT

C

DNA/PI

G2G2G1G1

SS

Cell cycle analysisCell cycle analysisBrdUrd/anti-BrdUrdBrdUrd/anti-BrdUrd

BrdUrd/anti-BrdUrdBrdUrd/anti-BrdUrd

Dynamic analysisDynamic analysis more complex procedure - denaturation of more complex procedure - denaturation of

DNADNA difficult to combine with another antibodydifficult to combine with another antibody

Exposure of the BrdUrdExposure of the BrdUrd

Denature DNA with 2 M HCl or heat Denature DNA with 2 M HCl or heat Partially digest DNA with Partially digest DNA with

endonuclease/exonucleaseendonuclease/exonuclease UV irradiation - label strand breaks with UV irradiation - label strand breaks with

Tdt/BrdUrd (SBIP)Tdt/BrdUrd (SBIP) Li et al., (1994) Int. J. Oncol., Li et al., (1994) Int. J. Oncol., 44, 1157., 1157.

UV irradiation in the presence of Hoechst 33258UV irradiation in the presence of Hoechst 33258 Hammers et al. (2000) Cytometry, Hammers et al. (2000) Cytometry, 4040, 327., 327.

Cell cycle analysisCell cycle analysisBrdUdr/anti-BrdUdrBrdUdr/anti-BrdUdr

Brd

Udr

/FIT

C

DNA/PI

0 h 4 h 8 h

G1G1

SS

G2G2

BrdUdr/anti-BrdUdrBrdUdr/anti-BrdUdr

Brd

Udr

/FIT

C

DNA/PI

0 h

G1G1

SS

G2G2

4 h 8 h

V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories)

Measurement of proliferationMeasurement of proliferation

BrdUrd/anti-BrdUrdBrdUrd/anti-BrdUrd

V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories)

Brd

Urd

/FIT

C

11

22

33

44

55

66

77

88

99

DNA

Window set in early to mid-S Window set in early to mid-S phasephase

Drug effects on cell cycleDrug effects on cell cyclepulse label after treatmentpulse label after treatment

Cells prepared in Institute for Cancer Studies, Sheffield

Incubated for 2 h with cisplatin 24 h earlier

No drug Drug

Nuclear & cytoplasmic Nuclear & cytoplasmic antigensantigens

Michael G. OrmerodMichael G. Ormerod

m.g.ormerod@btinternet.comm.g.ormerod@btinternet.com

Staining intracellular antigensStaining intracellular antigens

To detect intracellular antigens, the cells To detect intracellular antigens, the cells must be fixed or permeabilised. must be fixed or permeabilised.

Method used depends onMethod used depends on The antigen to be detectedThe antigen to be detected

The combination of stains used in a multi-The combination of stains used in a multi-parameter analysisparameter analysis

Staining intracellular antigensStaining intracellular antigens

The epitope on a particular antigen may be The epitope on a particular antigen may be sensitive to fixationsensitive to fixation

Consequently, there is no standard Consequently, there is no standard procedure for preparing cellsprocedure for preparing cells

A procedure has to be established for each A procedure has to be established for each new antibody.new antibody.

Fixatives for intracellular Fixatives for intracellular antigensantigens

Fixatives may be divided into two broad Fixatives may be divided into two broad classesclasses Those that cross-link proteins, such as Those that cross-link proteins, such as

paraformaldehydeparaformaldehyde Those that coagulate proteins and extract lipids, Those that coagulate proteins and extract lipids,

such as ethanol, methanol and acetonesuch as ethanol, methanol and acetone

The two may be combined - e.g. The two may be combined - e.g. paraformaldehyde followed by ethanolparaformaldehyde followed by ethanol

Permeabilisation of cellsPermeabilisation of cells

Unfixed cells can be permeabilised using a Unfixed cells can be permeabilised using a variety of detergents. These can be divided into variety of detergents. These can be divided into two classestwo classes Strong detergents, such as Triton-X 100, which will Strong detergents, such as Triton-X 100, which will

dissolve the plasma membrane on unfixed cellsdissolve the plasma membrane on unfixed cells Weak detergents, such as saponin, which will create Weak detergents, such as saponin, which will create

holes in the plasma membraneholes in the plasma membrane

Sometimes, cells are fixed and then Sometimes, cells are fixed and then permeabilisedpermeabilised

Procedures for intracellular Procedures for intracellular antigensantigens

Typical procedures include:Typical procedures include: Fixation in 70% ethanol at 0°CFixation in 70% ethanol at 0°C Fixation in absolute methanol at - 20°CFixation in absolute methanol at - 20°C Fixation in 1% paraformaldehyde followed by Fixation in 1% paraformaldehyde followed by

methanol, both at 0°Cmethanol, both at 0°C Incubation of fresh cells with antibody on ice in Incubation of fresh cells with antibody on ice in

the presence of detergent the presence of detergent For nuclear antigens, enucleation with a strong For nuclear antigens, enucleation with a strong

detergent followed by fixation.detergent followed by fixation.

Intracellular antigen + DNAIntracellular antigen + DNA

Either:Either: Fix in 70% ethanol at 0°C, orFix in 70% ethanol at 0°C, or Fix in 1% PFA followed by ethanol or methanolFix in 1% PFA followed by ethanol or methanol

Stain with antibody-FITCStain with antibody-FITC AnalyseAnalyse

Data supplied by W.E. Corver, Leiden

Labelled with anti-keratin 8/18 & PI

tumour

normal

diploid tumour?

Human ovarian CA ascitesHuman ovarian CA ascites

Fixed in PFA, methanol

CyclinsCyclins

Molt-4 cells. Data supplied by Frank Traganos, N Y.Molt-4 cells. Data supplied by Frank Traganos, N Y.

FIT

C

FIT

C

DNA

cyclin A

DNA

control Ig

Cyclin BCyclin B

W1L2 human lymphoblastoid cells

Ki-

S1 (

FIT

C)

DNA

vinblastine treated

Ki-

S1 (

FIT

C)

DNA

Isolated nucleiIsolated nuclei

Ki-S1, proliferation-related antigen.Nuclei from breast Ca cell line, ZR75.

G1G1

SSG2G2

MM

early early G1G1

Data supplied by Richard Camplejohn

Two antigens plus DNATwo antigens plus DNA

Fix the cellsFix the cells 1% paraformaldehyde at 0°C followed by methanol 1% paraformaldehyde at 0°C followed by methanol

at -20°Cat -20°C Stain for antigens using FITC & PEStain for antigens using FITC & PE Stain for DNAStain for DNA

Hoechst 3258Hoechst 3258 7-AAD7-AAD DRAQ5DRAQ5 PI + TO-PRO-3PI + TO-PRO-3

Cyclin B & p105 + Hoechst 33258Cyclin B & p105 + Hoechst 33258Prostate tumour cell line. Data supplied by James Jaccoberger.

Endoreduplication

mitotic cells

Further applications to cell and Further applications to cell and molecular biologymolecular biology

fluoresceinfluoresceinFDA

deaddead

fluoresceinfluorescein

viableviable

+ PI+ PI

Estimating cell viabilityEstimating cell viability Incubate with fluorescein diacetate (FDA) Incubate with fluorescein diacetate (FDA) Add propidium iodide (PI)Add propidium iodide (PI)

esteraseesterase

Estimating cell viabilityEstimating cell viability

live

dead

clumps

PIPI

fluo

resc

ein

fluo

resc

ein

Ovarian Ca Ovarian Ca cell line cell line labeled with labeled with FDA & PIFDA & PI

Monitoring electropermeabilisationMonitoring electropermeabilisation

Electroporate at 0°CElectroporate at 0°C Add propidium iodide (PI)Add propidium iodide (PI) Warm to 37°C and incubate 10 minWarm to 37°C and incubate 10 min Add fluorescein diacetate & incubate 10 Add fluorescein diacetate & incubate 10

minmin Analyse green and red fluorescenceAnalyse green and red fluorescence

Monitoring electropermeabilisationMonitoring electropermeabilisation

notnotpermeabilisedpermeabilised

permeabilisedpermeabilised

electroporateelectroporate + PI+ PI incubate 37°Cincubate 37°C add FDAadd FDA

fail to fail to resealreseal

resealreseal