Flow cytometry is a fast and stable method for the quantification of viable cells. Determining cell viability is a significant step when assessing a cell’s reply to medication or other environmental agents. 

It is additionally essential to recognize dead cells in a cell suspension in order to eliminate them from the study. Dead cells can produce artifacts as a consequence of non-specific antibody staining or by uptake of fluorescent probes. 

One way to test cell viability is by applying dye exclusion. Active cells possess membranes that are yet intact and reject different types of dyes that easily enter the damaged, permeable membranes of non-viable cells.

Aim

To distinguish between living and dead cells.

Principle of Viability staining using propidium iodide

Propidium Iodide (PI) can only pass through disordered areas of membrane of dead cells and intercalates with the DNA of the nuclei, emitting red fluorescence light.

Propidium iodide (PI) can not penetrate the membrane that is usually excluded from viable cells. PI binds to ds DNA by intercalating between base pairs. When Pi is excited at  488 nm, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm. 

For this characteristic feature, PI is used with other fluorochromes excited at 488 nm i.e fluorescein isothiocyanate (FITC) and phycoerythrin (PE).

Note: Propidium iodide is a suspected carcinogen and should be handled with care. The dye must be disposed of safely and in accordance with applicable local regulations.

Requirement

• Propidium iodide (PI) solution: Dissolve PI (Sigma, P 4170 or Invitrogen # P3566) in dH2O at 1 mg/ml. Store aliquots at -20C for up to 2 years. Aliquots that are frequently used can be stored at 4C for up to 2 months. Discard solutions of PI that have been exposed to room temperature for more than 48 hr, or if they appear dark red.
• PBS + 2% FBS or PBS + 0.1 % BSA
• FACS™ Tubes (5 mL round-bottom polystyrene tubes)
• Pipette Tips and Pipettes
• Centrifuge
• Vortex
• Flow Cytometry Staining Buffer or an equivalent solution containing BSA and sodium azide

Controls

• Negative control – cells without PI staining
• Positive control – cells stained with PI

Procedure

1. Harvest cells and aliquot up to 1 x 106 cells/100 μL into FACS tubes. Wash the cells by adding 2 mL PBS (or HBSS), centrifuging at 300 x g for 5 minutes and then decanting the buffer from pelleted cells. Repeat wash step for a total of 2 times.

Note: Staining of cell surface antigens with antibodies may be done at this point. PI cannot be used when labeling intracellular molecules.

2. Resuspend cells in 100 μL of Flow Cytometry Staining Buffer (Catalog # FC001).
3. To adjust flow cytometer settings for PI, add 5 – 10 μL of PI staining solution to a control tube of otherwise unstained cells. Mix gently and incubate for 1 minute in the dark.
4. Determine PI fluorescence (using the FL-2 or FL-3 channel) with a FACScan™ instrument.

Note: Use the FL-2 channel if staining only with PI. Collect PI fluorescence in the FL-3 channel if the cells have been stained with an FITC- or a PE-conjugated antibody.

5. Acquire data for unstained cells and single-color positive controls.
6. Add 5 – 10 µL of PI staining solution to each sample just prior to analysis. Set the stop count on the viable cells from a dot-plot of forward scatter versus PI.

Note: Do not wash cells after the addition of the PI staining solution.

References

• https://www.rndsystems.com/resources/protocols/flow-cytometry-protocol-analysis-cell-viability-using-propidium-iodide
• https://flowcyt.rutgers.edu/wp-content/uploads/2017/10/Cell-Viability-using-PI.pdf