View
223
Download
1
Category
Preview:
Citation preview
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
Recommended