1. Flow Cytometry at Boston University Medical Campus Introduction to some methods that we offer Yan Deng (X4-5225), ydeng@bu.edu Gerald Denis (X4-1371), gdenis@bu.edu

2. Flow cytometry simultaneously measures and analyzes multiple physical characteristics of single particles, usually cells, as they move in a fluid stream through a beam of light. Any suspended particle between 0.2 and 50μM is suitable. Larger particles, solid tissue or clumps of cells must be disaggregated to be analyzed. Examples: lymphocytes, protozoa, micron beads, chromosomes Definitions

3. The particles in the fluid stream scatter incident light, which reveals internal properties, size and granularity. The particles also fluoresce; they emit laser light at the interrogation point; this light is picked up by detectors arrayed at a different angle to detectors of scattered light. Definitions

4. fluidics optics electronics

5. Fluidics sample flow cell laser waste ~2 x 10 5 to 1 x 10 7 cells/ml sheath fluid

6. Electronics eventFSC (size) SSC (granularity) FL1 (green) FL2 (yellow) FL3 (red) FL4 etc 150030063884020- 22001002458550- 360080030070030- digitize

7. Fluidics Purpose of the fluidics system: 1.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 sheath fluid sample fluid ‘hydrodynamic focusing’ single file particles ● low flow rate ● narrow sample core ● high resolution ● high flow rate ● wide sample core ● low resolution

8. Fluidics Concerns 1.Shear rates for cells: check after you complete a run to ensure that the cells are intact. 2. Larger tips are needed for cell sorting.

9. Optics Excitation is accomplished by lasers that emit light at specified wavelengths. The atomic properties of the excitation media define this wavelength for a particular laser. The laser beam is focused on the sample core; lasers must be fixed in place. This is the most common and versatile wavelength for excitation of fluorochromes. Argon blue lasers emit light at 488 nm.

10. Fluorescein (FITC) Hoechst 33258Texas Red Propidium iodide (PI) Laser light must overlap with excitation wavelength

11. Optics 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

14. Excitation of Texas Red is not optimal with 488 nm laser

15. http://probes.invitrogen.com/resources/spectraviewer/ http://www.bdbiosciences.com/spectra/ For more information

16. Electronics The electronic system: 1.quantifies the voltage pulse 2.converts analog signals to digital values 3.performs compensation 4.transfers data to the computer for analysis.

17. Adjusting the voltage of the PMT helps to optimize capture of desired populations Electronics – Photomultiplier Tubes (PMTs)

18. Electronics - Amplifiers Amplifiers are of two types: linear or logarithmic Linear amplification is typically used with scatter. Logarithmic amplification is typically used with fluorescence. DNA content (Linear detection) DNA content (Log detection)

19. Electronics Threshold is the minimum pulse height above which a signal will be processed electronically.

20. Electronics When a threshold value is defined, only signals with intensities greater than or equal to the value are recorded. Adjustment of blood immunophenotype to exclude debris beforeafter

21. Fluorescence

22. Fluorescence intensity is proportional to the number of binding sites of the fluorescent compound, or “fluorochrome”. Remember, when labeling live cells, to keep the experiment at 4 0 C, because receptors are internalized and recycled, leaving their fluorochromes behind.

23. Immunophenotyping negative stain positive stain

24. Cluster of Differentiation antibody specificity CD3Pan-T lymphocytes CD4T helper/Inducer CD5T cells, subset B cells CD11bMonocytes, granulocytes, NK/T cells CD19B cells CD20B cells CD25 Il-2R, Tac IL-2 receptor, Activated T cells, B cells, NK cells, monocytes CD34 Progenitor cells CD69 Early activation antigen on T, B and NK cells

25. Immunophenotyping Two-color fluorescence of lymphocytes 48% 44% 7% 1% CD19-FITC CD3-PE Note the experiment-specific definition of the “negative” population

26. Fluorescence Multicolor analysis Spectral overlap

27. 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. UncompensatedOptimal

28. Compensation BeforeAfter Multicolor immunophenotyping

29. Data presentation formats

30. Gates

32. The uses of gates for cell sorting

33. Some applications Multiparameter sorting Cell cycle kinetics

34. Some applications DNA content for cell cycle / apoptosis Immune system activation