Authors
Creative Bioarray
Summary
Calcein AM is a widely used membrane-permeable cell marker that can permeate into intact cells. Once inside the cells, non-fluorescent calcein AM is hydrolyzed by intracellular esterases into the green fluorescent dye calcein, which is highly negatively charged and is well-retained in cell cytoplasm. Calcein AM has been used for studies of cell membrane integrity and for long-term cell tracking due to its low cellular toxicity. It has also been used to quantify the number of viable cells.
The calcein AM cell viability assay is an endpoint analysis method for cell viability. The fluorescent signal generated from the assay is proportional to the number of living cells in the sample. And the results can be obtained quickly, mostly within 2 hours.
Introduction
Plate cells in a microplate with the densities from 1,000 to 500,000 cells/mL. The range of cell concentrations will need to be optimized to ensure the best dynamic range.
Centrifuge at 250 x g for 5 minutes. Alternatively, transfer the cells to microfuge tubes for centrifugation and return to the plate to read.
Carefully discard the media supernate and add 100 μL of 1X Calcein AM DW Buffer.
Centrifuge at 250 x g for 5 minutes.
Remove the supernate and replace with 100 μL of fresh 1X Calcein AM DW Buffer.
Note: It is important to remove any carry-over media in the supernate, as phenol red and serum will interfere with the sensitivity of the assay.
Add 50 μL of freshly diluted Calcein AM Working Solution to each well.
Incubate for 30 minutes at 37°C.
Record fluorescence using a 490 nm excitation filter and a 520 emission filter.
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