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)

2. laser Forward light scatter FS detector blocker bar

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

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

5. Gating u Set a region on a histogram or cytogram u 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 time threshold

8. Changing the threshold setting debris

9. 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

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

11. Pulse shape analysis single cells two 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 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

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

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

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

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

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

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

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)

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

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)

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

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

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%

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

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

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

31. Pulse shape analysis signal time G1G2 2 x G1 laser flow 2x area htwidth

32. DNA peak DNA area clumps single Pulse shape analysis

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

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

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

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

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

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

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

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

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.

42. Cell cycle analysis BrdUdr/anti-BrdUdr BrdUdr/FITC DNA/PI 0 h 4 h 8 hG1 S G2

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

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

45. Window set in early to mid-S phase

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

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

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

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.

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

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

52. Procedures for intracellular antigens u Typical procedures include: l Fixation in 70% ethanol at 0°C l Fixation in absolute methanol at - 20°C l Fixation in 1% paraformaldehyde followed by methanol, both at 0°C l Incubation of fresh cells with antibody on ice in the presence of detergent l For nuclear antigens, enucleation with a strong detergent followed by fixation.

53. Intracellular antigen + DNA u Either: l Fix in 70% ethanol at 0°C, or l Fix in 1% PFA followed by ethanol or methanol u Stain with antibody-FITC u Analyse

54. Data supplied by W.E. Corver, Leiden Labelled with anti- keratin 8/18 & PI tumour normal diploid tumour? Human ovarian CA ascites Fixed in PFA, methanol

55. Cyclins Molt-4 cells. Data supplied by Frank Traganos, N Y. FITC DNA cyclin A DNA control Ig

56. Cyclin B W1L2 human lymphoblastoid cells

57. Ki-S1 (FITC) DNA vinblastine treated Ki-S1 (FITC) DNA Isolated nuclei Ki-S1, proliferation-related antigen. Nuclei from breast Ca cell line, ZR75. G1 S G2 M early G1 Data supplied by Richard Camplejohn

58. Two antigens plus DNA u Fix the cells l 1% paraformaldehyde at 0°C followed by methanol at -20°C u Stain for antigens using FITC & PE u Stain for DNA l Hoechst 3258 l 7-AAD l DRAQ5 l PI + TO-PRO-3

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

60. Further applications to cell and molecular biology

61. fluoresceinFDA dead fluorescein viable + PI Estimating cell viability u Incubate with fluorescein diacetate (FDA) u Add propidium iodide (PI) esterase

62. Estimating cell viability live dead clumps PI fluorescein Ovarian Ca cell line labeled with FDA & PI

63. Monitoring electropermeabilisation u Electroporate at 0°C u Add propidium iodide (PI) u Warm to 37°C and incubate 10 min u Add fluorescein diacetate & incubate 10 min u Analyse green and red fluorescence

64. Monitoring electropermeabilisation notpermeabilised permeabilised electroporate + PI incubate 37°C add FDA fail to reseal reseal