Cell counting and viability measurements are important for both clinical and research applications, including patient health diagnosis, growth rate studies, and any other study in which reagent amount depends on cell concentration. After speaking with experts from DeNovix, we have put together a list of seven tips to get the best results from your cell counting experiments.
No matter what you use for cell counting, the surface where you put the sample must be clean. Any contamination can get in the way of your cell counts leading to inaccurate results. For manual cell counting, this involves removing the glass coverslip, washing the counting chamber and coverslip with water or bleach, and drying with lab wipes or acetone. When using disposable slides, a new slide is required for each sample (or pair of samples if the slide has two chambers). In the case of the CellDrop Automated Cell Counter by DeNovix, the chamber is formed between two optical-grade sapphire surfaces arranged in parallel to each other. The sample is pipetted between the surfaces and is held in place by surface tension. To clean the surface, simply wipe it with a dry laboratory wipe.
Cell counting requires the analysis of a small sample of the whole stock solution, so care must be taken to ensure that the sample is representative of the original stock culture. While cell counters like the CellDrop may apply algorithms to identify cells within clumps, they cannot correct for a non-representative sample. Extracellular DNA and cell debris following cell lysis are common causes of clumping. Cell lysis may result from factors such as overgrowth, mechanical shearing through excessive pipetting, or freeze/thaw cycles, and under- or over-digestion with trypsin can also lead to heterogeneous samples. By avoiding the causes of cell lysis and filtering samples for cell debris, you can minimize the impact of cell clumps.
Another important step to ensure that the sample is representative is vortexing. When a solution is vortexed, the cells spread homogenously throughout the solution. Vortexing the solution immediately before loading the sample increases the probability that the sample will be homogenous. Adherent cells are particularly likely to form cell-to-cell aggregates and may benefit from being kept on a shaker while samples are prepared.
Whether you are counting by hand or letting an algorithm do it for you, it is important to adjust the focus and exposure settings to ensure the best possible visibility of cells for the most accurate results. The optimal focus and exposure will show a sharp contrast between the cell membrane and the background. For fluorescence applications, the ability to individually optimize each fluorescence channel will ensure the most reproducible results. Fluorescence intensities should be set to ensure that the cells are as bright as possible while remaining their true size—no light should bleed over the edges of the cells.
The CellDrop has a unique feature that allows for a wider range of cell density than other slide-based counters. The loading chamber height can be adjusted in the software according to the density of cells in your sample. On-screen guidance is given to ensure optimal height.
Most automated cell counters include settings that can be adjusted to best match your protocol. For example, if you are interested in cells of only a particular size, you can set a size range with minimum and maximum cell diameters. You can also set a roundness parameter that will make the software ignore debris. In the case of the CellDrop, it is possible to adjust the settings even after the cells have been counted, and the data will be reanalyzed according to the new parameters.
Brightfield analysis can be effective for counting cultured mammalian cells; however, it can be difficult to distinguish between live and dead cells using trypan blue, and for many primary cells, fluorescence is required. For example, peripheral blood mononuclear cells (PBMCs) are usually mixed with large numbers of red blood cells (RBCs) that may show up as “dead” using brightfield analysis. But with dyes such as acridine orange (AO) and propidium iodide (PI), it is possible to stain only PBMCs and to distinguish live from dead using the dual fluorescence capabilities of your cell counter. AO and PI both stain nucleic acids, but only AO is capable of crossing live cell membranes. As a result, live PBMCs are stained with AO and fluoresce green, dead PBMCs are stained with both AO and PI and fluoresce red, and RBCs are unstained and do not fluoresce at all.
Following these tips will help to improve your cell counting accuracy. For more information on the CellDrop Automated Cell Counter by DeNovix, which eliminates slide and plastic waste by using DirectPipette™, visit www.denovix.com/celldrop.