Cell Viability can be an important parameter to measure in flow cytometric analyses. It can be used as a quantitative parameter to assess the cytotoxicity of exogenous substrates or drugs, cytotoxic cellular interactions or as a method to eliminate dead cells from immunofluorescence analysis since non-viable cells may show different patterns of non-specific antibody binding compared to intact viable, competent cells. There are several methods that can be used to quantitate viability of cells. These methods typically use non-permeant dyes (e.g. propidium iodide, 7-Amino Actinomycin D) that do not enter cells with intact cell membranes or active cell metabolism. Cells with damaged plasma membranes or with impaired/no cell metabolism are unable to prevent the dye from entering the cell. Once inside the cell, the dyes bind to intracellular structures producing highly fluorescent adducts which identify the cells as "NON-VIABLE". Other methods can be used to assess viability by detection of active cell metabolism which can result in the conversion of a non-fluorescent substrate into a highly fluorescent product (e.g. fluorescein diacetate).

PROPIDIUM IODIDE (PI) is a non-permeant dye that can penetrate the membranes of dying/dead cells. It intercalates into the major groove of the DNA and produces a highly fluorescent adduct so NON-VIABLE cells can be identified by positive RED fluorescence. PI can be excited at 488 nm and its fluorescence detected from 550 up to 670 nm. Despite the broad emission spectrum of PI, it can be used in combination with other 488 nm-excited fluorochromes like FITC and PE but it is essential that proper compensation be used to ensure correct identification of PI(+) and PI(-) cells from those stained with the other fluorescent markers.

7-AMINO ACTINOMYCIN D (7-AAD) is another non-permeant dye that can be used to identify NON-VIABLE cells. (see Dave Coder's web page description). The 7-AAD is excited by the 488 nm laser line of an argon laser with fluorescence detected above 650 nm. Although the emission intensity of 7-AAD is lower than that of PI, the longer wavelength emission may make it more useful in combination with other 488 nm-excited fluorochromes such as FITC and PE.

FLUORESCEIN DIACETATE (FDA) is a non-polar, non-fluorescent fluorescein analogue which can pass through the cell membrane whereupon intracellular esterases cleave off the diacetate group producing the highly fluorescent product fluorescein. The fluorescein will accumulate in cells which possess intact membranes so the GREEN fluorescence can be used as a marker of cell viability. Cells which do not possess an intact cell membrane or an active metabolism may not accumulate the fluorescent product and therefore do not exhibit GREEN fluorescence. This may be used in combination with PI staining as the non-viable cells will take up the PI and stain RED whereas viable cells will not take up the PI and should only stain GREEN. This 2-colour separation of non-viable and viable cells provides for a more accurate quantitation of cell viability than single colour analysis.

A.

Protocol for PI viability:

1. Harvest cells and prepare single cell suspension in buffer (e.g. PBS + 2% FBS; PBS + 0.1% BSA).
2. Wash cells X2 and resuspend at 1-2 x 106 cells/ml.
3. Stain with monoclonal antibodies as required.
4. Resuspend in 1 ml staining buffer and assess autofluorescence signals for unstained cells. Establish PMT voltage settings for each fluorescence channel and set compensations for FITC and PE positive controls if needed.
5. Add 10 ml of PI staining solution to a separate tube of unstained cells, mix gently and incubate 1 min.
6. Determine PI fluorescence (FL2 or FL3 on the FACScan) and compensate signals accordingly. (Use FL2 if staining only with PI. If using FITC- and PE- labelled antibodies, collect PI fluorescence in FL3).
7. Acquire data for unstained cells, single-colour positive controls. Add PI to each subsequent sample prior to analysis and set the stop count on the VIABLE cells from a dot-plot of FSC vs PI.

REAGENTS:
Propidium Iodide Staining Solution:
10 mg/ml PI [Sigma, P 4170] in PBS.

B.

Protocol for 7-AAD viability:

1. Harvest cells and prepare single cell suspension in buffer (e.g. PBS + 2% FBS; PBS + 0.1% BSA).
2. Wash cells X2 and resuspend at 1-2 x 106 cells/ml.
3. Stain with monoclonal antibodies as required.
4. Resuspend in 1 ml staining buffer and assess autofluorescence signals for unstained cells.
5. Establish PMT voltage settings for each fluorescence channel and set compensations for FITC and PE positive controls if needed.
6. Add 10 ml of 7-AAD staining solution to a separate tube of unstained cells, mix gently and incubate 30 min. at 4oC in the dark.
7. Determine 7-AAD fluorescence (FL3 on the FACScan) and compensate signals accordingly.
8. Acquire data for unstained cells, single-colour positive controls. Add 7-AAD to each subsequent sample prior to analysis and set the stop count on the VIABLE cells from a dot-plot of FSC vs 7-AAD.

REAGENTS:
7-AAD Staining Solution:
1 mg/ml 7-AAD [Sigma, A 9400] in PBS.
Prepare stock 7-AAD 10mg/ml in DMSO and dilute 1:100 in PBS.

C.

Protocol for FDA and PI viability:

1. Harvest cells and prepare single cell suspension in buffer (e.g. PBS + 2% FBS; PBS + 0.1% BSA).
2. Wash cells X2 and resuspend at 1-2 x 106 cells/ml.
3. Add 10 ml of FDA working solution and 30 ml of PI working solution.
4. Incubate at room temperature for 3 min and place on ice until analysed.
5. Prepare single colour controls for compensation. Cells fixed in 1% paraformaldehyde can serve as your non-viable, positive control.

REAGENTS:
Propidium Iodide Stock Solution:
100 mg/ml PI [Sigma, P 4170] in PBS. Dilute 100 ml stock solution in 1 ml PBS for the working solution.

Fluorescein Diacetate Stock Solution:
5 mg/ml FDA [Sigma, F 7378] in ethanol. Dilute 40 ml stock solution in 10 ml PBS for working solution.