Every flow cytometry experiment, whether it begins in a culture flask or a mouse spleen, requires at least one step in which cells are counted. This step is essential to ensuring that you have a sufficient number of cells to evaluate by flow cytometry and that your staining and processing protocols are done correctly.
For decades, biologists have relied on counting cells using a special microscope slide called a hemocytometer. This slide holds fluid at a fixed volume and cells can be counted accurately by using a microscope. At present, automated cell counters are becoming more popular for their ease of use and rapidity. Both approaches can discriminate live cells from dead cells and can provide accurate cell counts when used correctly, but which approach is best for your experiment? Consider these factors when deciding how you want to count your cells.
Hemocytometers can be used to count almost any cell type and are well suited for accurately counting cells in heterogeneous samples as the end user can discriminated different cell types by eye. Automated cell counters count cells using a gating strategy and a computer algorithm, and as such are not as reliable for counting heterogeneous samples.
Number of Samples
Cell counting is typically needed when processing specimens for long term storage, such as PBMCs from peripheral blood. In situations where large numbers of samples are being processed, the rapid counting offered by an automated cell counter may be a critical time saving option compared with manual counting.
Hemocytometers rely on the use of a microscope for cell counting, but many automated cell counters are freestanding devices. Depending on your needs for portability, such as may be the case in biocontainment settings or in the field, an automated cell counter may be a better option.
Sometimes head-to-head comparisons using both approaches (Figure 1 above) may be the best way to decide which cell counting technique is best for your application.