Light scatter u Forward angle light scatter l in a narrow angle from the direction of the laser beam...

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Transcript of Light scatter u Forward angle light scatter l in a narrow angle from the direction of the laser beam...

  • Slide 1
  • Light scatter u Forward angle light scatter l in a narrow angle from the direction of the laser beam l FALS or FS u Right angle light scatter l at right angles to the laser beam l RALS or SS (side scatter)
  • Slide 2
  • laser Forward light scatter FS detector blocker bar
  • Slide 3
  • Light Scatter u The intensity of scatter is proportional to the size, shape and optical homogeneity of cells (or other particles) u It is strongly dependent on the angle over which it is measured l particularly with forward scatter
  • Slide 4
  • Light Scatter u Forward scatter tends to be more sensitive to the size and surface properties u can be used to distinguish live from dead cells u Side scatter tends to be more sensitive to inclusions within cells l can be used to distinguish granulated cells from non-granulated cells
  • Slide 5
  • Gating u Set a region on a histogram or cytogram u IF cell IN region THEN show another property
  • Slide 6
  • Cell selection by gating Gating on the lymphocytes. IF cell has light scatter in R1 THEN show on CD4/CD8 cytogram lymphocytes
  • Slide 7
  • Triggering the electronics signal time threshold
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  • Changing the threshold setting debris
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  • I distance cross-section spherical elliptical Shape of the laser beam Focus the laser beam with: l spherical lens - circular cross-section l cross cylindrical lens pair - elliptical X-section
  • Slide 10
  • Pulse shape analysis signal time laser flow Integrated area = total fluorescence Signal peak Signal width = beam width + cell diam.
  • Slide 11
  • Pulse shape analysis single cells two cells
  • Slide 12
  • DNA analysis by flow cytometry Michael G. Ormerod m.g.ormerod@btinternet.com
  • Slide 13
  • DNA content Ploidy Cell cycle
  • Slide 14
  • DNA Probes Use DNA probes that are stoichiometric - that is, the number of molecules of probe bound is equivalent to the quantity of DNA
  • Slide 15
  • Dyes for DNA cell cycle analysis u Propidium iodide l Excited at 488 nm; fluoresces red (617 nm) l easily combined with fluorescein stain l also stains RNA u DRAQ5 l Max. excitation at 646 nm; can be excited at 488 nm; fluoresces in deep red at 680 nm max l Taken up by live cells
  • Slide 16
  • Dyes for DNA cell cycle analysis u Hoechst dyes l excited by uv; fluoresce blue l DNA specific - bind to AT l Hoechst 33342 can be used to stain viable cells u DAPI l excited by uv; fluoresce blue l DNA specific
  • Slide 17
  • Definitions & Terms u DNA Ploidy l Related to the quantity of DNA in a cell u DNA Index l Ratio between the mean DNA content of the test cells to the mean DNA content of normal diploid cells, in G0/G1phase u Coefficient of Variation (CV) l 100*SD/mean DNA l Usually measured on G1/G0 cells
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  • FNA of human breast carcinoma PI stain DNA Cell number normal tumour G1 G2 S DNA content - measuring ploidy & SPF
  • Slide 19
  • DNA analysis of the cell cycle Following changes in the cell cycle
  • Slide 20
  • Quality control of DNA measurement u Sample preparation u Instrument alignment u Correct data analysis
  • Slide 21
  • Using propidium iodide for DNA analysis u Excited at 488 nm (argon-ion) u Fluoresces red u Does not cross intact plasma membrane l Permeabilise with detergent or l Fix in 70% ethanol or l Fix in paraformaldehyde followed by ethanol u Treat with RNase
  • Slide 22
  • Sample preparation for DNA analysis u Fixed cells l Samples can be stored l Needed when adding antibody stain l Quality may be reduced u Permeabilisied cells or nuclei l Use fresh or frozen samples, limited storage time l High quality achievable (Vindelov method)
  • Slide 23
  • DNA measurement Use linear amplification l Cell cycle is linear, not logarithmic l Relevant information occupies more of the histogram l Cell cycle algorithms assume a linear scale
  • Slide 24
  • Instrument alignment u Check daily using standard fluorescent beads u Correct alignment essential u (Some misalignment can be tolerated with immunofluoresence measurement - not DNA)
  • Slide 25
  • DNA measurement Use linear amplification l Cell cycle is linear, not logarithmic l Relevant information occupies more of the histogram l Cell cycle algorithms assume a linear scale
  • Slide 26
  • Quality control of DNA measurement Measure the spread of the distribution across the G1/G0 peak as coefficient of variation (cv)
  • Slide 27
  • C T D C, T cv = 1% D cv = 1.2% DNA measurement Human breast carcinoma cells prepared by the Vindelov method. PI stain. (Data supplied by Gyda Otteson & Ib Christensen, Finsen Laboratory, Copenhagen) AT C D C 1.2% T 1.0% D 1.0% A 2.2%
  • Slide 28
  • DNA histogram u Measure DNA content u Problem with clumps u 2 cells in G1 = 1 cell in G2 u Distinguish by pulse shape analysis
  • Slide 29
  • Shape of the laser beam focus with: l spherical lens - circular cross-section l cross cylindrical lens pair - elliptical cross- section I distance cross-section spherical elliptical
  • Slide 30
  • Flow Cytometry u Pulse shape analysis Integrated area = total fluorescence Signal peak Signal width = beam width + cell diam. PMT voltage time cell beam
  • Slide 31
  • Pulse shape analysis signal time G1G2 2 x G1 laser flow 2x area htwidth
  • Slide 32
  • DNA peak DNA area clumps single Pulse shape analysis
  • Slide 33
  • DNA width DNA area clumps single ungated gated Pulse shape analysis
  • Slide 34
  • Measuring cell proliferation using the BrdUrd/anti-BrdUrd method
  • Slide 35
  • Measuring cell proliferation u DNA histogram u BrdUrd/anti-BrdUrd u Hoechst/PI/BrdUrd u Dilution of label
  • Slide 36
  • DNA histogram u Static measurement of the cell cycle u First choice u Easy to combine with antibody stain
  • Slide 37
  • Cisplatin Following changes in the cell cycle Genotoxic drug S phase slow down G2 block
  • Slide 38
  • BrdUrd/anti-BrdUrd u Pulse label with BrdUrd (30 min) u Harvest cells at different times u Fix cells u Denature DNA (acid, heat or UV) u Label with anti-BrdUrd and PI
  • Slide 39
  • V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories) BrdUrd/FITC DNA/PI G2 G1 S Cell cycle analysis BrdUrd/anti-BrdUrd
  • Slide 40
  • BrdUrd/anti-BrdUrd u Dynamic analysis u more complex procedure - denaturation of DNA u difficult to combine with another antibody
  • Slide 41
  • Exposure of the BrdUrd u Denature DNA with 2 M HCl or heat u Partially digest DNA with endonuclease/exonuclease u UV irradiation - label strand breaks with Tdt/BrdUrd (SBIP) l Li et al., (1994) Int. J. Oncol., 4, 1157. u UV irradiation in the presence of Hoechst 33258 l Hammers et al. (2000) Cytometry, 40, 327.
  • Slide 42
  • Cell cycle analysis BrdUdr/anti-BrdUdr BrdUdr/FITC DNA/PI 0 h 4 h 8 hG1 S G2
  • Slide 43
  • BrdUdr/anti-BrdUdr BrdUdr/FITC DNA/PI 0 h G1 S G2 4 h8 h V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories) Measurement of proliferation
  • Slide 44
  • BrdUrd/anti-BrdUrd V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories) BrdUrd/FITC 1 2 3 4 5 6 7 8 9 DNA
  • Slide 45
  • Window set in early to mid-S phase
  • Slide 46
  • Drug effects on cell cycle pulse label after treatment Cells prepared in Institute for Cancer Studies, Sheffield Incubated for 2 h with cisplatin 24 h earlier No drugDrug
  • Slide 47
  • Nuclear & cytoplasmic antigens Michael G. Ormerod m.g.ormerod@btinternet.com
  • Slide 48
  • Staining intracellular antigens u To detect intracellular antigens, the cells must be fixed or permeabilised. u Method used depends on l The antigen to be detected The combination of stains used in a multi- parameter analysis
  • Slide 49
  • Staining intracellular antigens u The epitope on a particular antigen may be sensitive to fixation u Consequently, there is no standard procedure for preparing cells u A procedure has to be established for each new antibody.
  • Slide 50
  • Fixatives for intracellular antigens u Fixatives may be divided into two broad classes l Those that cross-link proteins, such as paraformaldehyde l Those that coagulate proteins and extract lipids, such as ethanol, methanol and acetone u The two may be combined - e.g. paraformaldehyde followed by ethanol
  • Slide 51
  • Permeabilisation of cells u Unfixed cells can be permeabilised using a variety of detergents. These can be divided into two classes l Strong detergents, such as Triton-X 100, which will dissolve the plasma membrane on unfixed cells l Weak detergents, such as saponin, which will create holes in the plasma membrane u Sometimes, cells are fixed and then permeabilised
  • Slide 52
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