Cell culture almost always includes a step where cells are counted. Whether the goal is to identify an appropriate seeding quantity for passage, harvest cells for downstream experiments, or monitor cell health, it is vital that cell counting is both accurate and efficient. Although hemocytometry has been used for decades to count cells, a growing awareness of its limitations has led many researchers to consider alternative methods. With modern Coulter counter-based systems offering quicker, easier, and more consistent cell counts than ever before—all within a convenient handheld device—could it be time that you and your hemocytometer said goodbye?
While hemocytometry is the most common way to count cells, being both widely accessible and relatively inexpensive, it is time-consuming and inherently prone to error. Briefly, during a typical hemocytometer-based cell count, a glass slide is placed over the counting chambers; 10 µL diluted cell sample (usually treated with a staining reagent such as Trypan Blue) is added to the hemocytometer using a pipette; cells are counted manually using a microscope; then a calculation is performed to obtain the cell concentration in cells/mL.
Each of these steps can be a major source of error. Inadequate placement of the glass slide and poor pipetting technique can both result in an incorrect volume being loaded into the hemocytometer, whereas counting cells manually is a further cause of inaccuracy—confusion often arises over whether to count cells that are touching the edges of the counting chamber fields, or whether cells should be classed as live or dead. The latter problem is compounded by the fact that many staining reagents can be cytotoxic if used inappropriately. Mistakes are also common during the calculation stage, with users frequently failing to account for the dilution factor, forgetting to divide by the number of counting chamber fields that were used, or multiplying by a value other than 104 to establish the final cell concentration.
Depending on the rationale for counting cells, the downstream impact of getting things wrong can vary considerably. Where cell counts are overestimated during passaging, the freshly seeded cell population may be too sparse to survive. In contrast, an underestimate of cell numbers during propagation can lead to overcrowding that may cause cells to undergo phenotypic change. Using too few cells for protein extraction, immunodetection, or a functional assay can mean the target of interest is undetectable or results are skewed. And an inaccurate assessment of cell health can lead to wasted time and resources when data is deemed unreliable and experiments must be repeated. For these reasons, many researchers prefer Coulter counting technology to hemocytometer-based methods for counting cells.
Coulter counters use electrical impedance to count particles, such as cells, in solution. As the sample is drawn through a small aperture between two electrodes, the change in electrical impedance provides an indication of cell numbers and size that is then automatically converted to a cell concentration. The advantages of Coulter counting include high sensitivity and the ability to detect a wide range of particle sizes; for example, this method enables the exclusion of small particles such as cell debris. Coulter counting also benefits from exceptional reproducibility since it rapidly counts extremely large numbers of cells and removes the effects of operator bias.
The size and expense of traditional Coulter counter instruments have historically discouraged researchers from obsoleting hemocytometer-based counts. Moreover, as well as taking up valuable bench space, many older Coulter counter models require specialized cleaning protocols, regular servicing by a qualified engineer, and often necessitate maintenance when tubing becomes blocked —all contributing factors preventing their more widespread adoption. However, with the development of modern, accessible, Coulter counter-based technologies for counting cells, hemocytometers may gradually be displaced as the most common cell enumeration tool.
Researchers can now count cells using a handheld portable device, generating results in seconds with no requirement for potentially harmful staining reagents and no need to transfer samples from the biosafety cabinet to the bench for counting. Easy-to-use modern systems use a precision-manufactured sensor to measure impedance as cells pass through microfluidic channels, displaying cell counts alongside a histogram of the overall population based on cell volume or diameter according to user preference. With data transferrable to a portable USB drive or via wireless transmission to a computer workstation for analysis and archiving, cell counting has never been easier or more accurate.
MilliporeSigma’s Scepter™ 3.0 is a compact handheld device for easy, highly accurate Coulter-based cell counting. To learn more, visit sigmaaldrich.com/scepter