About Calcein AM / PI Cell Viability Assays

The Calcein –AM/PI assay is a two-colour fluorescence cell death assay that is based on the simultaneous determination of live and dead cells with two different probes.

It measure recognized parameters of cell viability by evaluating intracellular esterase activity and plasma membrane integrity : calcein-AM and Propidium iodide (PI) .

The assay principles are general and applicable to most eukaryotic cell types, including adherent cells, suspension cells, and certain tissues, but not to bacteria or yeast.

The viable cells are distinguished by the presence of ubiquitous intracellular esterase activity, determined by the enzymatic conversion of the virtually nonfluorescent cell- permeant calcein AM to the intensely fluorescent calcein.

The calcein, a polyanionic dye is well retained within viable cells, producing an intense uniform green fluorescence in viable cells (ex/em ~495 nm/~515 nm).

Propidium iodide enters cells with damaged membranes in dead cells and undergoes a 40-fold enhancement of fluorescence upon binding to nucleic acids (DNA, RNA) thereby producing a bright red fluorescence in dead cells (ex/em ~535 nm/~617 nm).

Adopted from from BioRender

Cell Viability Assays Principle

PI is excluded by the intact plasma membrane of live cells.

The cell viability determination depends on these physical and biochemical properties of cells.

Cytotoxic events that do not affect these cell properties may not be accurately assessed using this method.

Background fluorescence levels are inherently low with this assay technique because the dyes are virtually non- fluorescent before interacting with cells.

This assay is suitable for use with a wide variety of techniques, including microplate assays immunocytochemistry, flow cytometry, and in vivo cell tracing

Cell Viability Assays Requirements

1. Calcein-AM fluorescent dye

2. Propidium iodide fluorescent dye

3. Phosphate Buffer Saline (PBS) (i.e. 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4.2H2O and 2 mM KH2PO4, pH 7.4).

4. Fluorescence microscope with blue (FITC) and green (Rhodamine) filters 5. 37oC water bath

Cell Viability Assays Procedure

1. Prepare the stock of Calcein-AM dye in DMSO at a concentration of 1mg/ml. Similarly prepare PI stock in sterile water at a concentration of 10mg/ml

NOTE: These dyes are highly light sensitive and should be stored in smaller aliquots frozen in -20`C till use; PI is a suspected mutagen and hence should be handled with great care

2. Adherent cells may be cultured on sterile glass coverslips as either confluent or subconfluent monolayers (e.g., fibroblasts are typically grown on the coverslip for 2–3 days until acceptable cell densities are obtained).

The cells may be then cultured inside 35 mm disposable petri dishes or other suitable containers; non-adherent cells may also be used.

NOTE: If inverted fluorescence microscope is used, then the cells can be grown in the culture plates and observed directly in the microscope If a normal (upright) fluorescence microscope is available, cells can be grown on coverslips, loaded with dye and carefully mounted on glass slides, and observed under microscope.

3. Remove the medium from the cells in the plates (for cells in suspension, centrifuge and remove the medium; for adherent cells medium can be directly removed).

4. Wash twice with PBS buffer. It is important to wash twice as traces of serum in the medium might interfere with the loading of the dyes.

5. Add fresh buffer and add Calcein-AM dye so that the final concentration of the dye is 1μg/ml.

6. Incubate at 37`C in dark for 15 minutes

7. Now add PI dye so that the final concentration is 10μg/ml. Incubate in dark at 37`C for a further 5 minute duration

8. Now remove the dye containing buffer and add fresh buffer.

9. Observe under the microscope FITC filter can be used for viewing the calcein stained live cells and Rhodamine filter can be used for PI stained dead cells

10. Count the number of calcein positive cells and PI positive cells also the total number of cells in each field of view. Count atleast five fields for each plate.

11. The cell viability can be expressed as (the number of calcein positive cells/ total number of cells) x 100