1. Page 1 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM BMS 631 - LECTURE 13 Flow Cytometry: Theory Bindley Bioscience Center Office: 765-494 0757 email; robinson@flowcyt.cyto.purdue.edu Cell Function J. Paul Robinson SVM Professor of Cytomics & Professor of Biomedical Engineering Purdue University WEB http://www.cyto.purdue.edu Notice: The materials in this presentation are copyrighted materials. If you want to use any of these slides, you may do so if you credit each slide with the author’s name. It is illegal to copy these to CourseHero or any other online theft system.

2. Page 2 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Cellular Response Cell death Cell ‘suicide’ Ignore damage Damage repair Incorrect repair

3. Page 3 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Functional Assays intracellular pH intracellular calcium intracellular glutathione oxidative burst phagocytosis

4. Page 4 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Oxidative Burst generation of toxic oxygen species by phagocytic cells superoxide anion measured with hydroethidine hydrogen peroxide measured with 2’,7’-dichlorofluorescin diacetate (DCFH-DA)

5. Page 5 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM TIME (seconds) 2400 1800 1200 600 Scale 345 115 38 12 4 Neutrophil Oxidative Burst PMA-Stimulated Neutrophils Unstimulated Neutrophils 0 Log DCF 1 10 100 1000

6. Page 6 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM FITC-Labeled Bacteria Phagocytosis

7. Page 7 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Cellular Functions Cell Viability Phagocytosis Organelle Function –mitochondria, ER –endosomes, Golgi Oxidative Reactions –Superoxide –Hydrogen Peroxide –Nitric Oxide –Glutathione levels Ionic Flux Determinations –Calcium –Intracellular pH Membrane Potential Membrane Polarization Lipid Peroxidation

8. Page 8 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Organelle Function MitochondriaRhodamine 123 EndosomesCeramides GolgiBODIPY-Ceramide Endoplasmic ReticulumDiOC 6 (3) Carbocyanine

9. Page 9 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Fluorescent Indicators How the assays work: Superoxide: Utilizes hydroethidine the sodium borohydride reduced derivative of EB Hydrogen Peroxide: DCFH-DA is freely permeable and enters the cell where cellular esterases hydrolyze the acetate moieties making a polar structure which remain in the cell. Oxidants (H 2 O 2 ) oxidize the DCFH to fluorescent DCF Glutathione: In human samples measured using 40  M monobromobimane which combines with GSH by means of glutathione-S-transferase. This reaction occurs within 10 minutes reaction time. Nitric Oxide: DCFH-DA can indicate for nitric oxide in a similar manner to H 2 O 2 so care must be used. DAF is a specific probe available for Nitric Oxide

10. Page 10 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Hydroethidine HE EB N CH 2 CH 3 NH 2 H2NH2N H Br - N CH 2 CH 3 NH 2 H2NH2N + O2-O2- Phagocytic Vacuole SOD H2O2H2O2 NADPH NADP O2O2 NADPH Oxidase OH - O2-O2- DCF HE O2-O2-O2-O2- H2O2H2O2H2O2H2O2 DCF Example: Neutrophil Oxidative Burst

11. Page 11 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM DCFH-DA DCFH DCF COOH H Cl O O-C-CH3 O CH3-C-O Cl O COOH H Cl OH HO Cl O COOH H Cl O HO Cl O Fluorescent Hydrolysis Oxidation 2’,7’-dichlorofluorescin 2’,7’-dichlorofluorescin diacetate 2’,7’-dichlorofluorescein Cellular Esterases H2O2H2O2 DCFH-DADCFH-DA DCFH DCF H O 2 2 2 2 Lymphocytes Monocytes Neutrophils log FITC Fluorescence.1.1 1000 100 10 1 0 20 40 60 counts PMA-stimulated PMN Control 80

12. Page 12 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Hydroethidine - Superoxide Production 15 minutes45 minutes

13. Page 13 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Endothelial Cell Oxidative Pathways Relative percentages of the mean intracellular EB fluorescence (O 2 - ) in rat pulmonary endothelial cells (REC) 60 min after stimulation with H 2 O 2. This figure is a summary of a number of possible oxidative pathways in REC. The Y axis shows a measurement of superoxide anion via oxidation of hydroethidine to ethidium bromide, as a percentage of the control (100%). XO mediated pathways are inhibited by nearly 50%. A combination of inhibitors of mitochondrial respiration, as well as solvents indicate the baseline oxidation of the probe (30-40%). (n=3)

14. Page 14 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Oxidative Reactions SuperoxideHydroethidine Hydrogen PeroxideDichlorofluorescein Glutathione levelsMonobromobimane Nitric OxideDichlorofluorescein

15. Page 15 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM 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

16. Page 16 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM 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

17. Page 17 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Membrane Polarization Polarization/fluidityDiphenylhexatriene How the assay works: The DPH partitions into liphophilic portions of the cell and is excited by a polarized UV light source. Polarized emissions are collected and changes can be observed kinetically as cells are activated. An image showing DPH fluorescence in cultured endothelial cells.

18. Page 18 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM CD16 Expression on Normal Cultured PMN 0 Hours negative control 48 Hours 24 Hours As cells age, the CD16 expression reduces The “bright” CD16 antigen is lost first

19. Page 19 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM PI - Cell Viability How the assay works: PI cannot normally cross the cell membrane If the PI penetrates the cell membrane, it is assumed to be damaged Cells that are brightly fluorescent with the PI are damaged or dead PI PI PI PI PI PI PI PI PI PI PI PI PI PI Viable CellDamaged Cell

20. Page 20 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Superoxide measured with hydroethidine Step 7C: Export data from Excel data base to Delta Graph Step 6C: Export data from measured regions to Microsoft Excel cell 1 cell 2 cell 3 cell 4 cell 5 Change in fluorescence was measured using Bio-Rad software and the data exported to a spread sheet for analysis. -200 0 200 400 600 800 1000 1200 1400 1600 1800 cell 1 cell 2 cell 3 cell 4 cell 5 Time in seconds 1000 1200 1400 1600 1800600 800 200 400 %change (DCF fluorescence)

21. Page 21 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Phagosome O2O2 O2-O2- H2O2H2O2 NADPH + H + NADP + HMP NADPH Oxidase GSSG GSH GR GP H2O2H2O2 SOD O2-O2- H+H+ H2OH2O Catalase H 2 O + O 2 PCB SOD PCB (Reduced GSH level) Stimulant PKC PCB (PMA) Human Neutrophil ? ? + O2-O2- OH. Lipid Peroxidation Phospolipase A2 activity Leukotrienes H2O2H2O2

22. Page 22 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Ionic Flux Determinations CalciumIndo-1 Intracellular pHBCECF How the assay works: Fluorescent probes such as Indo-1 are able to bind to calcium in a ratiometric manner The emission wavelength decreases as the probe binds available calcium Time (Seconds) 03672108144180 Stimulation 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) Flow CytometryImage Analysis

23. Page 23 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Light Scatter Changes of PMN at 24 Hours control lps bu ar

24. Page 24 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM Phagocytosis Uptake of Fluorescent labeled particles Determination of intracellular or extracellular state of particles How the assay works: Particles or cells are labeled with a fluorescent probe The cells and particles are mixed so phagocytosis takes place The cells are mixed with a fluorescent absorber to remove fluorescence from membrane bound particles The remaining fluorescence represents internal particles FITC-Labeled Bacteria

25. Page 25 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM 1 2 3 3 2 1 405/35 nm 460 nm Calcium ratioing study with Indo-1 Changes in the fluorescence were measured using the Bio-Rad calcium ratioing software. The same region in each wave length was measured and the relative change in each region was recorded and exported to a spread sheet for analysis.. Export data from measured regions to Microsoft Excel Export data from Excel data base to Delta Graph 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 cell 1 cell 2 cell 3 Ratio: intensity1 (460nm) / intensity2 (405/35nm) http://www.cyto.purdue.edu

26. Page 26 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM On Calcofluor White No warranties on this one but Calcofluor White M2R (Fluorescent brightener28, Sigma) may work but staining may not be very specific. Lectins are another possible alternative. A staining technique for differentiating starch granules and cell walls was developed for computer- assisted studies of starchgranule distribution in cells of wheat (Triticum aestivum L.)caryopses. Blocks of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by using stains prepared in 80% ethanol and using short staining times. The IKI quenched background fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell area was similar for both wheat classes. The starch granule sizes ranged from 2.1 microns 3 to 22,000 microns 3 with approximately 90% of the granules measuring less than 752 microns 3 (ca.11 microns in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was measured for four cells near the central cheek region of a single caryopsis. The percentage of small granules was higher at the ends than at the mid-section of the cells Source: From: Richard Haugland (richard.haugland@probes.com) Date: Thu Feb 07 2002 - 17:04:48 ESTrichard.haugland@probes.com http://www.cyto.purdue.edu/hmarchiv/current/1041.htm References: Biotech Histochem 1992 Mar;67(2):88-97 Block-surface staining for differentiation of starch and cell walls in wheat endosperm. Glenn GM, Pitts MJ, Liao K, Irving DW. Western Regional Research Center, USDA-ARS, Albany, California 94710.

27. Page 27 ©1990-2013 J.Paul Robinson, Purdue University 12:02 AM About SNARF-1 SNARF®-1 carboxylic acid, acetate, succinimidyl ester http://www.probes.com/servlets/product?region=Select&item=2 2801 is a very new probe that we have not yet tested for assessing cell cycle. http://www.probes.com/servlets/product?region=Select&item=2 2801 We have tested it for labeling cells and for cell tracing. I am not aware of any publications that have used it for that, however. However, it requires the same hydrolysis of the acetates as does CFSE and has the same succinimidyl ester as CFSE and should therefore have similar utility and have its fluorescence decrease by half on cell division, as does CFSE. Its potential advantage is that it can be excited at 488 nm but has red fluorescence so it may be complementary to CFSE. Source: From: Richard Haugland (richard.haugland@probes.com) Date: Thu Jan 17 2002 - 20:10:07 ESTrichard.haugland@probes.com http://www.cyto.purdue.edu/hmarchiv/current/0872.htm

28. Page 28 ©1990-2013 J.Paul Robinson, Purdue University Summary There are a variety of functional probes useful in flow cytometry Many require live cells for the entire assay period Timing for kinetic assays is critical You must match the probe to the excitation as usual 12:02 AM