1. Current Research Applications of Flow Cytometry and Cell J.Paul Robinson Professor of Immunopharmacology Professor of Biomedical Engineering Purdue University Email: jpr@flowcyt.cyto.purdue.edujpr@flowcyt.cyto.purdue.edu WEB: http://www.cyto.purdue.edu Faculty Lecture at Kitasato University, Towada, Japan June 26-July 4, 2000

2. Lecture summary This lecture will discuss the principles of flow cytometry and how they are applied to basic research and clinical questions. We will discuss the general principles of how a flow cytometry operates and why this technology has advantages over many others. In addition, we will look as some examples of newer applications such as apoptosis, multiplexed bead assays and future applications. Cell sorting using your recently acquired Coulter Altra will be described and the key features discussed.

3. Purdue University Cancer Center & Purdue University Cytometry Laboratories

4. What can Flow Cytometry Do? Enumerate particles in suspension Determine “biologicals” from “non-biologicals” Separate “live” from “dead” particles Evaluate 10 5 to 10 6 particles in less than 1 min Measure particle-scatter as well as innate fluorescent Measure 2 o fluorescence Sort single particles for subsequent analysis

5. Introductory Terms and Concepts Parameter/Variable Light Scatter- Forward (FALS), narrow (FS) - Side, Wide, 90 deg, orthogonal Fluorescence - Spectral range Absorption - loss of light Time - Kinetics Count- number of events/particles/cells

6. Concepts Scatter: Size, shape, granularity, polarized scatter (birefringence) Fluorescence: Intrinsic: Endogenous pyridines and flavins Extrinsic: All other fluorescence profiles Absorption: Loss of light (blocked) Time: Useful for kinetics, QC Count: # events -always part of any collection

7. Instrument Components Electronics: Control, pulse collection, pulse analysis, triggering, time delay, data display, gating, sort control, light and detector control Optics: Light source(s), detectors, spectral separation Fluidics: Specimen, sorting, rate of data collection Data Analysis: Data display & analysis, multivariate/simultaneous solutions, identification of sort populations, quantitation

8. Arc Lamp Excitation Spectra Irradiance at 0.5 m (mW m -2 nm -1 )         Xe Lamp Hg Lamp Shapiro p 99

9. Lasers Argon laser He-Ne Laser

10. Optical Collection systems He-Cd Laser Argon Laser He-Ne Laser 2 nd Argon Laser

11. Elite Cytometer with 4 Lasers Water cooled argon laser He-Cd laser Air-cooled argon laser Santa clause

12. Optical Design PMT 1 PMT 2 PMT 5 PMT 4 Dichroic Filters Bandpass Filters Laser Flow cell PMT 3 Scatter Sensor Sample

13. Coulter Optical System – Elite/Altra The Elite optical system uses 5 side window PMTs and a number of filter slots into which any filter can be inserted 555 - 595 PMT4 APC 655 - 695 PMT6 PMT7 4 9 0 D L 488 BK 0 5 5 D L 6 2 5 D L 675 BP 488 BP525 BP575 BP Purdue Cytometry Labs PUCL3034 632 BP TM PMT3 PMT2 PMT1 PMT5

14. Fluidics

15. SMALL BEADLARGE BEAD Frequency Histogram SMALL BEAD LARGE BEAD Sample in Sheath Sheath in Laser beam Stream Charge +4KV -4KV Waste SORT RIGHT SORT LEFT SORT DECISIONS Piezoelectric crystal oscillator Last attached droplet LEFT RIGHT Sensors Sensor Signals are collected from several sensors placed forward or at 90° to the laser beam. It is possible to “sort” individual particles. The flow cell is resonated at a frequency of approximately 32KHZ by the piezoelectric crystal mounted on the flow cell. This causes the flowing stream to break up into individual droplets. Gating characteristics can be determined from histograms (shown right) and these can be used to define the sort criteria. These decisions are all controlled by the computer system and can be made at rates of several thousand per second. Figure 1 The central component of a flow cytometer is the flow cell. A cutdown of a typical flow cell indicates the salient features. Sample is introduced via the sample insertion rod. Sheath fluid (usually water or saline) is ntroduced to surround the insertion rod causing hydrodynamic focussing of flowing cells which are contained within a core fluid. The laser intersects the fluid either outside the flowcell (in air) or in a slightly extruded portion of the flow cell tip (in quartz).

16. Fluorescence The wavelength of absorption is related to the size of the chromophores Smaller chromophores, higher energy (shorter wavelength)

17. Fluorescence Stokes Shift –is the energy difference between the lowest energy peak of absorbance and the highest energy of emission 495 nm 520 nm Stokes Shift is 25 nm Fluorescein molecule Fluorescnece Intensity Wavelength

18. Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm 457350514610632488 Common Laser Lines

19. Fluorescence Resonance Energy Transfer Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 2

20. Flow cytometry measurements L M G SCATTER FLUORESCENCEIMAGE

21. Data Presentation Formats Histogram Dot plot Contour plot 3D plots Dot plot with projection Overviews (multiple histograms)

22. Data Analysis Concepts Gating Single parameter Dual parameter Multiple parameter Back Gating Note: these terms are introduced here, but will be discussed in more detail in later lectures

23. FITC Fluorescence Mo1 CD4 CD8 CD45 leu11a CD20 Tube ID

25. The Cell Cycle G1G1 M G2G2 S G0G0 Quiescent cells

26. A DNA histogram G 0 -G 1 S G 2 -M Fluorescence Intensity Cell Number

27. A typical DNA Histogram G 0 -G 1 S G 2 -M Fluorescence Intensity # of Events

28. log Thiazole Orange.1 1000 100 10 1 RMI = 0 log Thiazole Orange.1 1000 100 10 1 RMI = 34 Reticulocyte Analysis

29. Labeling Strand Breaks with dUTP [Fluorescein-deoxyuridine triphosphate (dUTP)] Green Fluorescence is Tdt and biotin-dUTP followed by fluorescein-streptavidin Red fluorescence is DNA counter-stained with 20µg/ml PI PI-Red Fluorescence Green Fluorescence Side Scatter Forward Scatter Green: apoptotic cells Red: normal cells R2: Apoptotic Cells R1: Normal Cells

30. Scatter Pattern of Human leukocytes Lymphocytes Monocytes Neutrophils A flow cytometry scattergram Forward scatter (size) Side scatter (granularity)

31. Three Color Lymphocyte Patterns CD3 CD4 CD3 CD4 CD8 Data from Dr. Carleton Stewart

32. YoYo-1 stained mixture of 70% ethanol fixed E.coli cells and B.subtilis (BG) spores. mixture BG E.coli BG E.coli mixture Run on Coulter XL cytometer Scatter Fluorescence Scatter

33. Live cell/dead cell PI Fluorescence Data from Dr. Doug Redelman, Sierra Cytometry PI Hoechst 33342

34. Oxidative Reactions SuperoxideHydroethidine Hydrogen PeroxideDichlorofluorescein Glutathione levelsMonobromobimane Nitric OxideDichlorofluorescein

35. Calcium Flux 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 050100150200 Ratio: intensity of 460nm / 405nm signals Time (seconds) Time (Seconds) 03672 108 144180 Stimulation Flow CytometryImage Cytometry

36. Membrane Potential Oxonol Probes Cyanine Probes How the assay works: Carbocyanine dyes released into the surrounding media as cells depolarize Because flow cytometers measure the internal cell fluorescence, the kinetic changes can be recorded as the re-distribution occurs Time (sec) Green Fluorescence Repolarized Cells 05121024 0 300 150 012002400 Time (sec) 05121024 Green Fluorescence PMA Added fMLP Added Depolarized Cells

37. Summary Main Applications DNA and RNA analysis Phenotyping Cell Function Sorting and cell isolation Immunological assays

38. The facilities at Kitasato University

39. Coulter Altra The facilities at Kitasato University

40. Coulter XL Cytometer The facilities at Kitasato University