2. Flow Cytometry Basics: What, How and Why?

3. KAM TJU FCM-09 -1.0SciGro, Inc. Flow Cytometry: The 30,000 Foot View What?  High speed, low resolution measurement of the properties of individual cells (or other particles) entrained in a flowing liquid (usually saline). How?  A flow cytometer is an instrument that detects and measures single cells/particles in suspension as they pass through a sensing region  Use of fluorescent probes allows different biological properties of a cell to be quantified based on its interaction(s) with varying wavelengths of light. Why?  Faster, more sensitive and more quantitative measurement of cell properties than fluorescence microscopy  Multicolor single cell analysis allows subpopulations present in a mixture to be characterized without first physically isolating them  Even very rare cells can readily be detected  In flow cytometers with sorting capability, selected subpopulations of cells with defined properties can be physically retrieved for study

4. Flow cytometry basics: How?  Instrumentation laserslasers fluidicsfluidics opticsoptics electronicselectronics sortingsorting  Data display and analysis Single parameter (“histograms”)Single parameter (“histograms”) Multiparameter (“dot plots”, “contour plots”)Multiparameter (“dot plots”, “contour plots”) Listmode data and gatingListmode data and gating  Fluorescence measurements Excitation and emissionExcitation and emission FluorochromesFluorochromes FiltersFilters Color compensation (correction for color overlap)Color compensation (correction for color overlap) KAM TJU FCM-08-1.0 SciGro, Inc.

5. KAM TJU FCM-09 -1.0SciGro, Inc. HOW NOT TO BE A FLOW CYTOMETRIST Drawing by Ben Givan

6. KAM TJU FCM-09 -1.0SciGro, Inc. Flow Cytometry vs. Microscopy PMT 1 PMT 2 LASER DETECTORS LIGHT SOURCE Adapted from figure kindly provided by Dr. Frank Mandy (Health Canada) Low throughput, high resolutionHigh throughput, low resolution

7. KAM TJU FCM-09 -1.0SciGro, Inc. LASER PMT 1234 Flow Cytometry: Single Cell Analysis in a Fluid Stream Figure courtesy of Dr. Frank Mandy (Health Canada)

8. KAM TJU FCM-09 -1.0SciGro, Inc. Why Use Flow Cytometry?  Can readily detect discrete and/or rare subpopulations  Can quantify differences between subpopulations without physical separation  Can be combined with flow sorting, to allow physical isolation and characterization of specified subsets

9. KAM TJU FCM-09 -1.0SciGro, Inc. Starting Material: Stained Cells Cultured U937 cell stained with propidium iodide What a Flow Cytometer Can (and Cannot) Tell You 2 Hours at 37º C.18 Hours at 37º C. Monocytes stained with fluorescein-tagged antibody to Fc  R1 (green fluorescence) and re-stained with phycoerythrin- tagged antibody to Fc  R1 (red fluorescence) after varying incubation times to allow receptor internalization 0 Hours at 37º C. Photomicrographs courtesy of Dr. Paul Wallace (RPCI)

10. KAM TJU FCM-09 -1.0SciGro, Inc. What Does A Flow Cytometer Look Like? (10,000 Foot View…) Basic ingredients Fluidics to deliver stream of particles single file to the analysis point where light beam is focused Light source & focusing optics Filters, detectors & electronics to detect particle- light interaction and convert to digital format Computer to record and analyze digitized data Block diagram courtesy of John Martin (Los Alamos Nat’l. Flow Cytometry Resource)

11. KAM TJU FCM-09 -1.0SciGro, Inc. What Does A Flow Cytometer Look Like? (Up Close and Personal…) Lasers Fluorescence & right angle light scatter pickup lens Quartz flow cell & sensing region Forward light scatter detector Photograph courtesy of John Martin (Los Alamos Nat’l. Flow Cytometry Resource) “Jet in air” (most common configuration for instruments that both analyze and sort cells) Glass flow cell (most common configuration for instruments that do high sensitivity analysis but not cell sorting)

12. KAM TJU FCM-09 -1.0SciGro, Inc. What Happens When a Cell Interacts With Light? (It Depends…!) Sources of cellular fluorescence Intrinsic: naturally occurring fluors – e.g. flavins, chlorophyll, other “autofluorescent” cellular components Extrinsic: deliberately by the experimenter to reflect different biological or biochemical properties – e.g. DNA binding dyes, antibodies, etc.) Figure adapted from J. Treadway, (Quantum Dot Corp.) Light is elastically scattered in all directions (no change in color/ wavelength) Scatter at low angles from the exciting light beam is a function of cell size and refractive index Scatter at high angles is a function of surface “roughness” or cytoplasmic granularity Interaction of exciting light and dye(s) or naturally occurring cell components leads to fluorescence Emitted light differs in color from exciting light (longer wavelengths) Emitted intensity a function of number of dyes per cell

13. KAM TJU FCM-09 -1.0SciGro, Inc. Why Use Fluorescence?

14. KAM TJU FCM-09 -1.0SciGro, Inc. Cell Interrogation Laser Beam Shaping Lens Quartz Flow Cell Figure courtesy of Dr. Paul Wallace (RPCI)

15. KAM TJU FCM-09 -1.0SciGro, Inc. SS PMT Laser FS Photodiode Quartz Flow Cell 500 LP Dichroic Mirror 530 bp Filter 560 SP Dichroic Mirror FL2 PMT FL1 PMT 580 bp Filter Q: HOW Do Flow Cytometers “See” Colors? A: Through wavelength-selective filters Figure courtesy of Dr. Paul Wallace (RPCI)

16. KAM TJU FCM-09 -1.0SciGro, Inc. Block diagram courtesy of John Martin (Los Alamos Nat’l. Flow Cytometry Resource) Basic ingredients Fluidics to deliver stream of particles to analysis point where light beam is focused Light source & focusing optics Filters, detectors & electronics to detect particle- light interaction and convert to digital format Computer to record and analyze digitized data Getting Inside the Black Box: How Are Cell/Light Interactions Converted to Digital Data?

17. KAM TJU FCM-09 -1.0SciGro, Inc. Photodetectors: Turning Light into Electrons Input Scattered laser light Fluorescence (multiple colors) Output Electric current (10 -6 – 10 -9 amps) (Emits electrons when hit by light) (Amplify photoelectrons; voltage dependent) (Electron avalanche catcher!) Figures courtesy of John Martin (Los Alamos Nat’l. Flow Cytometry Resource)

18. KAM TJU FCM-09 -1.0SciGro, Inc. PMT Quart Flow Cell Laser Condenser time pulse height 1000 0 200 345 862 List Mode File u Width or time u Area under the curve u Peak Turning Electrons into Digital Pulses: Step 1 Figure courtesy of Dr. Paul Wallace (RPCI)

19. KAM TJU FCM-09 -1.0SciGro, Inc. Using hardwired circuitry (older flow cytometers, slower electronics) Figures courtesy of John Martin (Los Alamos Nat’l. Flow Cytometry Resource) Cell Enters beam Cell Exits Beam Using digital signal processing (newer flow cytometers, fast electronics) Cell Enters beam Cell Exits Beam Turning Electrons into Digital Pulses: Step 2

20. KAM TJU FCM-09 -1.0SciGro, Inc. Block diagram courtesy of John Martin (Los Alamos Nat’l. Flow Cytometry Resource) Getting Inside the Black Box… What Does the Data Look Like? Getting Inside the Black Box… What Does the Data Look Like? Basic ingredients Fluidics to deliver stream of particles single file to the analysis point where light beam is focused Light source & focusing optics Filters, detectors & electronics to detect particle- light interaction and convert to digital format Computer to record and analyze digitized data

21. KAM TJU FCM-09 -1.0SciGro, Inc. Raw FCS Data File (untranslated) F $ Figure courtesy of A. Givan

22. KAM TJU FCM-09 -1.0SciGro, Inc. FCS Data file (translated) Event # Parameter value (channel #, proportional to signal intensity) Figure courtesy of A. Givan

23. KAM TJU FCM-09 -1.0SciGro, Inc. Do It Yourself Cytometry (in-class exercise)

24. KAM TJU FCM-09 -1.0SciGro, Inc. PMT 1 LASER Single Parameter Data Display: Frequency Histogram Number of events Channel Number [low][medium][high] Adapted from figure kindly provided by Dr. Frank Mandy (Health Canada) 256512768 [Relative Fluorescence Intensity] 11024

25. KAM TJU FCM-09 -1.0SciGro, Inc. Reporting the Data: Single Parameter Histograms Measures of peak location/signal intensity 1) Modal Channel Number (most commonly observed value) 2) Median Channel Number (equal # of events above and below) 3) Mean Channel Number (average value; for Mean Fluorescence Intensity often abbreviated MFI) Measures of peak width/signal heterogeneity 1) Standard deviation (SD) 2) Coefficient of Variation (CV) = relative standard deviation = SD/mean x 100 Mode: a = b; a, b > c Median:a = b; a, b < c Mean:a = b = c SD, CV: c >> a > b

26. KAM TJU FCM-09 -1.0SciGro, Inc. Single Color Immunofluorescence Analysis % Positive 48 %12 % Adapted from figure kindly provided by A. Givan (Raw channel numbers using 4 decade log amps) M1 = analysis Marker, region 1 for FL1 ( dim – bright FL1) KAM TJU FCM-08-1.0 SciGro, Inc. M1 = analysis Marker, region 1 for FL2 ( dim – bright FL2)

27. KAM TJU FCM-09 -1.0SciGro, Inc. Two Parameter Data : 1+1 = 4 (not 2) A B A B A B + + + + ---- NOTE: ++ events could be both colors on one cell OR 2 single color cells stuck together Figure courtesy of Dr. Frank Mandy (Health Canada)

28. KAM TJU FCM-09 -1.0SciGro, Inc. Two-color data display (dot plot) and analysis (quadrant statistics) Figure courtesy of A. Givan Note logarithmic scale display showing relative fluorescence intensities (not raw channel numbers)

29. KAM TJU FCM-09 -1.0SciGro, Inc. Different Types of Two-Parameter Data Plots “Dot” Plot “Contour” Plot “3D” Plot “Density” Plot Figure courtesy of A. Givan KAM TJU FCM-08-1.0 SciGro, Inc. Signal Intensity 

30. KAM TJU FCM-09 -1.0SciGro, Inc. Side Light Scatter (SS) Forward Light Scatter (FS) Lymphocytes Monocytes Basophils Granulocytes Two Kinds of Light Scatter: Leukocyte Differential? Eosiniphils Neutrophils Mid 1980’s Figure courtesy of Dr. Frank Mandy (Health Canada)

31. KAM TJU FCM-09 -1.0SciGro, Inc. Light Scattering Properties Reflect Intrinsic Differences Among Cells (and Viewers) PLOT IT ANY WAY YOU LIKE! FSC = forward or “low angle” scatter; strongly influenced by cell size SSC = side or “right angle” scatter; reflects granularity or internal structure “Beckman Coulter” display“Becton Dickinson” display Lymphocytes Monocytes Granulocytes RBCs, debris Granulocytes Figure courtesy of A. Givan KAM TJU FCM-08-1.0SciGro, Inc. Signal Intensity 

32. KAM TJU FCM-09 -1.0SciGro, Inc. Light Scatter: It’s All Relative (Same sample, two different amplifier settings) Figure courtesy of A. Givan

33. KAM TJU FCM-09 -1.0SciGro, Inc. Electronic “Gating”: The Power Behind Flow Cytometry Figure courtesy of A. Givan

34. KAM TJU FCM-09 -1.0SciGro, Inc. Flow Essentials 04.04.17 (p. 33) Light Scatter Gating In Action Listmode Data File (translated) 2P Dot Plots FS:300-610 SS:50-400 Figure courtesy of Dr. Paul Wallace (RPCI) G1 gate values: G1

35. KAM TJU FCM-09 -1.0SciGro, Inc. Light Scatter Gating: Differentiating Among Leukocytes (no physical separation required) Figure courtesy of A. Givan KAM TJU FCM-08-1.0 SciGro, Inc.

36. KAM TJU FCM-09 -1.0SciGro, Inc. Fluorochromes for Antibody Tagging: Choice, choices…. FITC488525 PE (Phycobiliprotein) 488575 APC (Phycobiliprotein) 630650 PerCP ™ (Phycobiliprotein) 488680 Cascade Blue360450 Coumarin350450 Texas Red ™ 610630 Tetramethylrhodamine 550575 CY3 (indotrimethinecyanines) 540575 CY5 (indopentamethinecyanines) 640670 ProbeExcitationEmission Courtesy of J. Paul Robinson, Purdue Univ. Cytometry Facility

37. KAM TJU FCM-09 -1.0SciGro, Inc. Useful References and Resources FLOW CYTOMETRY  Melamed, M.R., Lindmo, T. and Mendelsohn, M.L., Eds., Flow Cytometry and Sorting, Second Edition, Wiley-Liss (1990).  Darzynkiewicz, Z., Robinson, J.P. and Crissman, H.A., Eds., Flow Cytometry, Parts A and B (Methods in Cell Biology, Volumes 41 and 42), Academic Press (1994).  Shapiro, H.S. Practical Flow Cytometry (4th Ed.), Wiley-Liss (2003); read-only version available online at http://probes.invitrogen.com/products/flowcytometry/practicalflowcytometry.html.  Robinson, J.P., Ed., Current Protocols in Cytometry, John Wiley and Sons (1997).  Givan, A.L., Flow Cytometry: First Principles (2 nd Ed), Wiley-Liss (2001).  http://www.isac-net.org/ (International Society for Advancement of Cytometry home page; especially links for “Presentations/Lectures”, “Purdue Discussion” and “Jobs/Positions” http://www.isac-net.org/  http://www.cytometry.org/ (Clinical Cytometry Society home page) http://www.cytometry.org/  http://wiki.clinicalflow.com/ (Clinical flow wiki, sponsored by DeNovo Software) http://wiki.clinicalflow.com/  http://flowbook-wiki.denovosoftware.com/ (wiki version of “Flow Cytometry – A Basic Introduction” by M.G. Ormerod, sponsored by DeNovo Software) http://flowbook-wiki.denovosoftware.com/  http://www.vsh.com/cytometrycourses/ (listing of applications-focused workshops and courses, sponsored by Verity Software House) http://www.vsh.com/cytometrycourses/