by K Chico, Jessica Piczon

The calcein AM (calcein-acetoxymethyl ester) assay is a widely used fluorescent technique for monitoring cell viability, chemotaxis, cell adhesion and multidrug resistance. When cells in culture are exposed to calcein AM, it passes the plasma membrane and is hydrolyzed by cytosolic unspecific esterases to become fluorescent calcein in living cells. Upon analysis, fluorescence can be directly or indirectly correlated to cell number, and therefore viability, within a sample. In general, analysis either measures the absolute or relative differences in fluorescent frequencies within a sample.

Absolute measurements can assess the number of cells within a sample directly. Absolute measurements do not require a reference standard or previously known absorbance data and are especially useful for samples which absorb and emit in wavelength ranges for which there are no reliable standards available. First, the cell number of the sample must be determined. Then, wells in the plate are diluted, then duplicated or triplicated appropriately.

Next, the calcein AM assay can be performed as normal and analyzed under a microplate reader. The average of each replicate value can then be determined, and the data can be plotted as a function of cell number per well versus fluorescence intensity. This method calculates all the photons emitted during fluorescence by each well, so it is especially important that the microplate reader is calibrated and that adequate gain adjustments are made beforehand.

Alternatively, relative measurements can be achieved using a comparative method. Here, the cell number of a test sample is calculated by comparing its fluorescence intensity to another reference sample with a known fluorescence. One benefit of obtaining relative measurements is that it is not necessary to calibrate the microplate reader beforehand since data is presented as the percent change in fluorescence intensity relative to an experimental control. Though this is more common than absolute measurements, the relative method does, however, require knowledge of the absorbance of both the reference and the sample.

It is important to note that this approach is also only applicable to samples in a solution because the measurement requires knowledge of the refractive index of the solvent, and the absorbance of both the reference and sample. Analysis relies on the use of well-characterized reference standards, with known fluorescence values and optical properties that closely match the sample of interest.

In procedure, wells in the plate are diluted, then duplicated or triplicated appropriately alongside a reference. Next, the calcein AM assay can be performed as normal and analyzed under a microplate reader. In analysis, the integrated fluorescence intensity of the reference is compared against the sample to subsequently calculate its fluorescence. Then, the ratio of fluorescence of the two solutions can then be correlated to the cell number within the test sample.