Yan Deng (X4-5225), ydeng@bu

1. Introduction to the Principles of Flow Cytometry for users of the Flow Cytometry Core Facility at BUMC 21 September 2010 Mike Xie (X4-5225), mike.xie@bmc.org Yan Deng (X4-5225), ydeng@bu.edu John Meyers (X8-7543), johnm@bu.edu Gerald Denis (X4-1371), gdenis@bu.edu

2. Flow Cytometry Core Facility: Personnel Director of Cell Sorting Services: Yanhui Deng Operations Manager: Mike Xie Facility Consultant: John Meyers Co-Director/Outreach: Gerald Denis Director: David Sherr http://www.bu.edu/cores/flow-cytometry

3. Where are the instruments located? FACScan: R901 FACScan, FACSCalibur:L508 U R here FACScan: X620 LSR II, MoFlo:670-5 http://www.bu.edu/cores/flow-cytometry

5. Flow cytometry was not easy in the old days ungloved hands missing PPE gendered division of labor live anthrax bacilli

6. But today… it’s as easy as PCR.

7. 3 elements of any flow cytometry system fluidics optics electronics

8. fluidics

9. ● low flow rate ● high flow rate ● narrow sample core ● wide sample core ● high resolution ● low resolution sample fluid Fluidics sheath fluid • Purpose of the fluidics system: • Transport particles in a fluid stream • to the laser beam to be interrogated • 2. Position the sample core in the center • of the laser beam ‘hydrodynamic focusing’ single file particles Always filter your samples to remove aggregates.

10. Fluidics When conditions are right (i.e. when turbulence is minimal): sample fluid flows in a central core does not mix with the sheath fluid This is termed ‘laminar flow’

11. optics electronics “SSC” “FSC”

12. Forward and side scatter of leukemic cells Normal B cells Malignant, large B cells SSC FSC The intensity of forward scatter light is proportional to size and cross-sectional area of the cells. The intensity of side scatter light is proportional to size, shape and internal structure/irregularity of the cells.

13. Photomultiplier (PMT) detectors convert photons (selected by mirrors and filters) to electrical pulses Peak Height (volts) Peak Area Peak Width (time) The higher the PMT voltage (user controllable), the greater the output magnitude for a given photon. At higher PMT voltages, the level of noise will also increase.

14. Photomultiplier Tubes (PMTs) FSC: forward scatter (size; cross-sectional area) SSC: side scatter (granularity, internal or surface structure that scatters light) Adjusting the voltage of the PMT helps to optimize capture of desired populations

15. A dotplot represents two properties of a single cell SSC FSC

16. A histogram represents the distribution of a single parameter across many cells 10,000 cells each! ControlCondition Experimental 1 cell 1 cell 1 cell 1 cell

17. DNA content (Linear detection) DNA content (Log detection) Electronic processing of emission signals Amplifiers are of two types: linear or logarithmic Linear amplification is typically used with scatter. Logarithmic amplification is typically used with fluorescence.

18. Gating Gating allows one to select populations based on computer or human-derived criteria and further gate or display the included cells

19. Backgating – don’t lose your bearings! Backgating allows one to determine if a gating strategy is all-inclusive of a desired cell type. In the above example, some cells are missed! What are they? Many investigators overlook the importance of verification by backgating!

20. FLUORESCENCE blue laser Excitation wavelength and emission wavelength are unique properties of each specific molecular structure (FITC)

21. Stokes Fluorescence Excitation Emission Stokes shift

22. Laser light must overlap with excitation wavelength Propidium iodide (PI) Fluorescein (FITC) 488 488 ex em yes ex em Hoechst 33258 Texas Red 488 488 no ex em ex em

23. Widely-used molecules are excited by the 488 nm laser (FACScan) 488 nm (Blue) : FITC, GFP, PE, PerCP, PE-Cy5, PI, PerCP-Cy5.5, PE-Cy7 But different lasers are available to excite other molecules (LSR II) 355 nm (UV) : Indo-1, DAPI, Alexa Fluor 350, Hoechst 33258 405 nm (Violet) : Alexa Fluor 430, Alexa Fluor 405,Pacific Blue 561 nm (Yellow/Green): Texas Red, Cherry Red, Tomato Red 633 nm (Red): APC, APC-Cy7, Alexa Fluor 647, Alexa 680

24. Octagon Detector Arrays emitted fluorescent light longpass dichroic mirrors bandpass filters

25. EMISSION

26. Fluorescence detection autofluorescence strongly expressed epitope weakly expressed epitope Note logarithmic scale

27. Isotype controls How do you know it’s real? weakly expressed epitope isotype control test Isotype controlantibodies should be used at the same concentration to stain cells at the same cell density as the experimental, but they give fluorescent signals that define a negative result.

28. Resolution sensitivity Resolution sensitivity, the ability to resolve a faint signal from background) depends on the difference D between the positive and background peaks and the spread of the background peak W

29. Choose the right fluor for the job! ReagentStain Index Phycoerythrin (PE) 356.3 Alexa Fluor 647 313.1 APC 279.2 PE-Cy7 278.5 PE-Cy5 222.1 PerCP-Cy5.5 92.7 PE-Alexa Fluor 610 80.4 Alexa Fluor 488 75.4 FITC 68.9 PerCP 64.4 APC-Cy7 42.2 Alexa Fluor 700 39.9 Pacific Blue 22.5 AmCyan 20.2 i.e. pick a bright fluor for a dim epitope and avoid spillover of bright cell populations into detector channels that require high sensitivity for rare signals

30. Problems in Emission Fluorescence Excitation Emission Spectral overlap

31. Filters resolve overlapping wavelengths of emitted light Longpass filter: transmits light of longer than or equal to a specific wavelength Shortpass filter: transmits light of shorter than or equal to a specific wavelength Bandpass filter: transmits light only within a narrow range of wavelengths Optical solutions to spectral overlap: Filters

32. Examples of optical filters in flow cytometry

33. Optical detector configurations red trigon octagon APC bandpass 660/20 longpass 735 LP bandpass 780/60 APC-Cy7

34. EMISSION two bandpass filters

35. Uncompensated Optimal Electronic solutions to spectral overlap: Compensation To correct for emission spillover of FITC signal (normally detected in the FL1 channel) into the FL2 channel (which detects PE), it is necessary to use filters or electronic compensation or both.

36. COMPENSATION Multicolor immunophenotyping Before After

37. No antibody. Autofluorescence only. No compensation applied.

38. CD4-PE. No compensation applied.

39. CD4-PE. Correct compensation applied. 1.4% PE subtracted from FITC PMT, 6.5% PE subtracted from APC PMT.

40. CD8-FITC. No compensation applied.

41. CD8-FITC. Correct compensation applied. 12.5% FITC subtracted from PE PMT.

42. CD4-PE + CD8-FITC. Streptavidin-APC alone.

43. CD4-PE + CD8-FITC. CD3-biotin + Streptavidin-APC 3 colors, correctly compensated

44. Spectral overlap of some fluorochrome combinations cannot be compensated easily or at all APC Cy5 Therefore, avoid such combinations

45. Contour plots provide more accurate data representation than dot plots

46. Gates granularity → size →

47. The uses of gates for cell sorting SSC FSC CD8 CD3

48. Four Applications Cell cycle analysis Multicolor immunophenotyping Phosphoprotein and kinase signaling Stem cell sorting by the “side population” method

49. Cell cycle analysis G0/G1 S G2/M S anti-BrdU-FITC (DNA synthesis) G2/M G0/G1 2N 4N 2N 4N DNA content (propidium iodide) 7-aminoactinomycin D Linear detection

50. 100 100 80 80 60 60 % of Max % of Max 40 40 20 20 0 0 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 100 100 80 80 60 60 % of Max % of Max 4 10 40 40 3 20 20 10 0 0 2 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 1 10 0 10 0 200 400 600 800 1000 Multicolor, Auto Compensation with Flow Jo isotype isotype gate 1: lymphocytes gate 2: B cells gate 6: T cells? FITC Pacific Blue isotype isotype etc gates 3 – 5 up to 11 colors PE PE-Cy7