Cell counting is an integral part of determining cell concentrations for plating in culture, determining cell viability, and assessing the results of cell isolation procedures
. It is recommended to perform an initial cell count prior to cell isolation; this number can then be compared to the cell count after cell isolation to calculate cell recovery. Additionally, viable cell counts should be performed when a decrease in cell viability may be expected, for example, when working with cryopreserved cells
or cells manipulated ex vivo.
Cell counting can be performed using Trypan Blue
or 3% Acetic Acid with Methylene Blue
. When performing a total nucleated cell count, 3% Acetic Acid with Methylene Blue is recommended. Acetic acid lyses the cellular membranes, and the methylene blue stains the exposed nuclei. Because mature red blood cells lack nuclei, they are excluded when counting. Alternatively, Trypan Blue is recommended for counting viable mammalian cells. Trypan Blue penetrates the cell membrane, thus it enters the cytoplasm of cells with compromised membranes (dead cells) to stain them blue. The live cells remain intact and can be distinguished from dead cells by their ability to exclude the blue dye. In this case one would count the intact viable cells.
This protocol describes how to perform total nucleated cell counts with 3% Acetic Acid with Methylene Blue, and how to perform viable cell counts by Trypan Blue dye exclusion. Additonal cell counting resources and templates
to help streamline your assays are also available.