1. PROPIDIUM IODIDE EXCLUSION ASSAY A rapid and reliable method to quantify viable cells in a suspension 2. Determination of cell viability is critical when evaluating the response to cytotoxic drugs or other environmental factors. In addition, it is often necessary to detect dead cells in a cell suspension in order to exclude them from the analysis. Dead cells can generate artifacts as a result of nonspecific antibody binding or through unwanted uptake of fluorescent probes. PURPOSE: 3. PROPIDIUM IODIDE PI -- intercalating agent fluorescent molecule Molecular mass -- 668.4 Da Can be used to stain cells 4. PI is a membrane impermeant dye -- generally excluded from viable cells Penetrate cell membranes of dying or dead cells Binds to double stranded DNA by intercalating between base pairs When PI does gain access to nucleic acids and intercalates -- fluorescence increases dramatically -- therefore used to identify dead cells Stained cells are determined Flow cytometery Flourescent microscopy 5. FACS Tubes (5 mL round-bottom polystyrene tubes) Pipette Tips and Pipettes Centrifuge Vortex MATERIALS REQUIRED: 6. PBS (1X): 0.137 M NaCl, 0.05 M NaH2PO4, pH 7.4 or Hanks Balanced Salt Solution (HBSS; 1X) Flow Cytometry Fixation Buffer (R&D Systems, Catalog # FC004, or an equivalent solution containing BSA and sodium azide) PI Staining Solution: 10 g/ml PI in PBS stored at 4 C in the dark Detection Antibodies (optional) Isotype Control Antibodies (optional) REAGENTS REQUIRED 7. Harvest cells and aliquot up to 1 x 106 cells/100 L into FACS tubes. Wash the cells 2 times by adding 2 mL of PBS, centrifuging at 300 x g for 5 minutes, and then decanting the buffer from the pelleted cells. Resuspend cells in 100 L of Flow Cytometry Staining Buffer. Add 5 - 10 L of PI staining solution. Mix gently and incubate for 1 minute in the dark. Determine PI fluorescence with a FACScan instrument. Note: Do not wash cells after the addition of the PI staining solution. PROCEDURE: 8. ADVANTAGES Quick and inexpensive Simple, rapid and reliable method for assessing viable cells Only a small fraction of total cells from a population is required 9. TRYPAN BLUE ASSAY A rapid and reliable method to quantify viable cells in a suspension 10. PRINCIPLE Dye binds to intracellular proteins of leaky cells. 11. procedure: Incubation with trypan blue dye. Live cells can not take up because of intact cell membranes. Only dead cells are stained. Visualization: Light microscope Automated cell counter and analyzer e.g; Cedex XS Analyzer or Cedex HiRes Analyzer. 12. ADVANTAGES: Viable cells can be counted Quick and inexpensive. Small sample size is required. 13. DISADVANTAGES: Every sample must be counted individually. Subjective evaluation with hemocytometer is a limitation. Not detection of apoptosis. Detects only necrotic cells. 14. LIMITATIONS Each individual sample must be counted; only a few tests may be simultaneously performed. Not specific for apoptosis PI is a suspected carcinogen and should be handled with care. The dye must be disposed of safely and in accordance with applicable local regulations.