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Light scatter

Light scatter

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Light scatter

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  1. Light scatter • Forward angle light scatter • in a narrow angle from the direction of the laser beam • FALS or FS • Right angle light scatter • at right angles to the laser beam • RALS or SS (side scatter)

  2. FS detector laser blocker bar Forward light scatter

  3. Light Scatter • The intensity of scatter is proportional to the size, shape and optical homogeneity of cells (or other particles) • It is strongly dependent on the angle over which it is measured • particularly with forward scatter

  4. Light Scatter • Forward scatter tends to be more sensitive to the size and surface properties • can be used to distinguish live from dead cells • Side scatter tends to be more sensitive to inclusions within cells • can be used to distinguish granulated cells from non-granulated cells

  5. Gating • Set a region on a histogram or cytogram • IF cell IN region THEN show another property

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

  7. Triggering the electronics signal threshold time

  8. Changing the threshold setting debris

  9. I spherical distance cross-section elliptical Shape of the laser beam Focus the laser beam with: • spherical lens - circular cross-section • cross cylindrical lens pair - elliptical X-section

  10. signal time Pulse shape analysis flow laser Integrated area = total fluorescence Signal peak Signal width = beam width + cell diam.

  11. Pulse shape analysis two cells single cells

  12. DNA analysis by flow cytometry Michael G. Ormerod m.g.ormerod@btinternet.com

  13. DNA content Ploidy Cell cycle

  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

  15. Dyes for DNA cell cycle analysis • Propidium iodide • Excited at 488 nm; fluoresces red (617 nm) • easily combined with fluorescein stain • also stains RNA • DRAQ5 • Max. excitation at 646 nm; can be excited at 488 nm; fluoresces in deep red at 680 nm max • Taken up by live cells

  16. Dyes for DNA cell cycle analysis • Hoechst dyes • excited by uv; fluoresce blue • DNA specific - bind to AT • Hoechst 33342 can be used to stain viable cells • DAPI • excited by uv; fluoresce blue • DNA specific

  17. Definitions & Terms • DNA Ploidy • Related to the quantity of DNA in a cell • DNA Index • Ratio between the mean DNA content of the test cells to the mean DNA content of normal diploid cells, in G0/G1phase • Coefficient of Variation (CV) • 100*SD/mean DNA • Usually measured on G1/G0 cells

  18. FNA of human breast carcinoma PI stain Cell number DNA DNA content - measuring ploidy & SPF G1 tumour normal G2 S

  19. DNA analysis of the cell cycle Following changes in the cell cycle

  20. Quality control of DNA measurement • Sample preparation • Instrument alignment • Correct data analysis

  21. Using propidium iodide for DNA analysis • Excited at 488 nm (argon-ion) • Fluoresces red • Does not cross intact plasma membrane • Permeabilise with detergent or • Fix in 70% ethanol or • Fix in paraformaldehyde followed by ethanol • Treat with RNase

  22. Sample preparation for DNA analysis • Fixed cells • Samples can be stored • Needed when adding antibody stain • Quality may be reduced • Permeabilisied cells or nuclei • Use fresh or frozen samples, limited storage time • High quality achievable (Vindelov method)

  23. DNA measurement Use linear amplification • Cell cycle is linear, not logarithmic • Relevant information occupies more of the histogram • Cell cycle algorithms assume a linear scale

  24. Instrument alignment • Check daily using standard fluorescent beads • Correct alignment essential • (Some misalignment can be tolerated with immunofluoresence measurement - not DNA)

  25. DNA measurement Use linear amplification • Cell cycle is linear, not logarithmic • Relevant information occupies more of the histogram • Cell cycle algorithms assume a linear scale

  26. Quality control of DNA measurement Measure the spread of the distribution across the G1/G0 peak as coefficient of variation (cv)

  27. Human breast carcinoma cells prepared by the Vindelov method. PI stain. (Data supplied by Gyda Otteson & Ib Christensen, Finsen Laboratory, Copenhagen) D C, T cv = 1% C T A D cv = 1.2% D C T DNA measurement C 1.2% T 1.0% D 1.0% A 2.2%

  28. DNA histogram • Measure DNA content • Problem with clumps • 2 cells in G1 = 1 cell in G2 • Distinguish by pulse shape analysis

  29. Shape of the laser beam focus with: • spherical lens - circular cross-section • cross cylindrical lens pair - elliptical cross-section I spherical distance cross-section elliptical

  30. Flow Cytometry • Pulse shape analysis Integrated area = total fluorescence cell beam Signal peak Signal width = beam width + cell diam. PMT voltage time

  31. flow width Pulse shape analysis G1 G2 2 x G1 laser 2x signal ht area time

  32. Pulse shape analysis single DNA peak clumps DNA area

  33. ungated DNA width gated DNA area Pulse shape analysis clumps single

  34. Measuring cell proliferation using the BrdUrd/anti-BrdUrd method

  35. Measuring cell proliferation • DNA histogram • BrdUrd/anti-BrdUrd • Hoechst/PI/BrdUrd • Dilution of label

  36. DNA histogram • Static measurement of the cell cycle • First choice • Easy to combine with antibody stain

  37. S phase slow down G2 block Following changes in the cell cycle Cisplatin Genotoxic drug

  38. BrdUrd/anti-BrdUrd • Pulse label with BrdUrd (30 min) • Harvest cells at different times • Fix cells • Denature DNA (acid, heat or UV) • Label with anti-BrdUrd and PI

  39. BrdUrd/anti-BrdUrd Cell cycle analysis V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories) S BrdUrd/FITC G2 G1 DNA/PI

  40. BrdUrd/anti-BrdUrd • Dynamic analysis • more complex procedure - denaturation of DNA • difficult to combine with another antibody

  41. Exposure of the BrdUrd • Denature DNA with 2 M HCl or heat • Partially digest DNA with endonuclease/exonuclease • UV irradiation - label strand breaks with Tdt/BrdUrd (SBIP) • Li et al., (1994) Int. J. Oncol., 4, 1157. • UV irradiation in the presence of Hoechst 33258 • Hammers et al. (2000) Cytometry, 40, 327.

  42. BrdUdr/anti-BrdUdr 8 h 4 h 0 h Cell cycle analysis BrdUdr/FITC S G2 G1 DNA/PI

  43. BrdUdr/anti-BrdUdr 4 h 8 h Measurement of proliferation 0 h S BrdUdr/FITC G1 G2 DNA/PI V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories)

  44. 4 1 7 8 5 2 3 9 6 BrdUrd/anti-BrdUrd V79 cells (data supplied by G. D. Wilson,, CRC Gray Laboratories) BrdUrd/FITC DNA

  45. Window set in early to mid-S phase

  46. No drug Drug Drug effects on cell cyclepulse label after treatment Incubated for 2 h with cisplatin 24 h earlier Cells prepared in Institute for Cancer Studies, Sheffield

  47. Nuclear & cytoplasmic antigens Michael G. Ormerod m.g.ormerod@btinternet.com

  48. Staining intracellular antigens • To detect intracellular antigens, the cells must be fixed or permeabilised. • Method used depends on • The antigen to be detectedThe combination of stains used in a multi-parameter analysis

  49. Staining intracellular antigens • The epitope on a particular antigen may be sensitive to fixation • Consequently, there is no standard procedure for preparing cells • A procedure has to be established for each new antibody.

  50. Fixatives for intracellular antigens • Fixatives may be divided into two broad classes • Those that cross-link proteins, such as paraformaldehyde • Those that coagulate proteins and extract lipids, such as ethanol, methanol and acetone • The two may be combined - e.g. paraformaldehyde followed by ethanol