How do scientists identify types of cells? 1. Morphologically (RBC, granulocyte, lymphocyte)

1. How do scientists identify types of cells? • 1. Morphologically (RBC, granulocyte, lymphocyte) • 2. Functionally • 3. Phenotypically • Use of monoclonal antibodies directed against • cell surface proteins • Can also use certain chemicals (PNA, antigen, PI) • Involves some sort of “visible” label • radioactivity • enzyme-substrate reaction (histochemistry) • fluorescence

2. Fluorescence Chemical or protein that absorbs light at one wave length (excitation) and emits light at a second wave length (emission) First must know properties of flurochrome you wish to use

3. 350 457 488 514 610 632 300 nm 400 nm 500 nm 600 nm 700 nm Common Laser Lines PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid Robinson et al. Purdue Cytometry Laboratory

4. Propidium Iodide Emisson Excitation 300 nm 400 nm 500 nm 600 nm 700 nm 400 nm 500 nm 600 nm 700 nm DNA PI

5. Fluorescein (FITC) Emisson Excitation 300 nm 400 nm 500 nm 600 nm 700 nm Wavelength 400 nm 500 nm 600 nm 700 nm Protein

6. Phycoerytherin (PE) Emisson Excitation 300 nm 400 nm 500 nm 600 nm 700 nm Protein

7. Allophycocyanin (APC) Protein 632.5 nm (HeNe) 300 nm 400 nm 500 nm 600 nm 700 nm Excitation Emisson

8. Flurochromes are generally stimulated by laser beams • (single wavelength of light). • 1. Fluorescent microscope (very simple to very • complex) • tissues or single cells • 2. Flow cytometer - single cells in suspension • Optics • Fluids • Electronics • Which to use?

9. Typical Clinical Cytometer Detector & Mechanical Computer System Fluidics Robinson et al. Purdue Cytometry Laboratory

12. Flow Cytometry Optics PMT 4 PMT Dichroic 3 Filters Flow cell PMT 2 Bandpass Filters PMT 1 Laser Robinson et al. Purdue Cytometry Laboratory

13. Optical Filters Dichroic Filter/Mirror at 45 deg Light Source Transmitted Light Reflected light

14. Standard Band Pass Filters 630 nm BandPass Filter White Light Source Transmitted Light 620 -640 nm Light

15. Standard Long Pass Filters 520 nm Long Pass Filter Light Source Transmitted Light >520 nm Light Standard Short Pass Filters 575 nm Short Pass Filter Light Source Transmitted Light <575 nm Light

16. Injector Tip Sheath fluid Fluorescence signals Focused laser beam Fluidics-Flow Cell Robinson et al. Purdue Cytometry Laboratory

17. Electronics • PMT collect light signal • Converted to electronic signal • Interpreted by software (Cell Quest) • Color and intensity on each cell

18. What can we measure? • Light scatter • forward = size • side = granularity • Fluorescence • Time • Ratio Robinson et al. Purdue Cytometry Laboratory

19. Laser FALS Sensor 90LS Sensor Light Scatter Robinson et al. Purdue Cytometry Laboratory

20. Cellular Functions • Cell Viability • Phagocytosis • Organelle Functions • mitochondria, ER • endosomes, golgi • Oxidative reactions • superoxide • hydrogen peroxide • nitric oxide • glutathione levels • Ionic Flux • Calcium • Intracellular pH • Membrane potiential • Membrane polorization • Lipid peroxidation Robinson et al. Purdue Cytometry Laboratory

21. TypicalAssays • Immunophenotyping • DNA content, cell-cycle analysis, ploidy • Chromosome karyotyping • Phagocytosis • Kinetic assays • Intracellular protein measurements • Total protein Robinson et al. Purdue Cytometry Laboratory

22. Research uses of flow cytometery Drug discovery Toxicity testing Cell culture studies Functional studies Immunology Hematology Pathology Microbiology Genetics Chemical engineering Biotechnology Agronomy Animal Science Robinson et al. Purdue Cytometry Laboratory

23. General Protocol for Immunophenotyping 1. Make single cell suspension 2. Deplete red blood cells if necessary 3. Count cells 4. Add blocking reagent to block FC binding 5. Add dyes/mAbs 6. Incubate on ice 7. Wash out unbound dye 8. Analyze

24. Y Anti-CD4 FITC Y Y Y Y No Fc blocking Direct => B cells look positive! Correct!!!

25. Indirect Streptavidin-flurochrome Labeled 2nd antibody Y Y Biotin-mAb Y Y Y Y Y Y Biotin-avidin 1 =goat anti-mouse CD4 2 =sheep anti-goat Ig-FITC

26. What does flow cytometry data look like? Histogram Number of cells Dye

27. Dot plot Only dye 2 Both dyes Dye 2 No dye Only dye 1 Dye 1

28. + Fluorescence Activated Cell Sorting FALS Sensor 488 nm laser Fluorescence detector - Charged Plates Single cells sorted into test tubes