Compared to manual, hemocytometer-based methods for counting cells, automated cell counters increase both consistency and throughput for more reliable results. For this reason, they have become an essential technology for many researchers, especially those working with limited primary cell material. Primary cells are notoriously difficult to count reproducibly as samples often contain large quantities of cellular debris. Moreover, primary cell viability is readily compromised by the length of time taken to manually perform the multiple counts necessary to achieve statistical relevance. This article discusses some of the advantages of automating cell counting and describes how an automated cell counter with dual fluorescence and brightfield optics streamlined a study using murine primary cells to investigate a potential immuno-oncology drug target.
Manual, hemocytometer-based cell counting typically uses Trypan Blue to stain dead cells within a suspension, enabling researchers to perform a live/dead cell count with a brightfield microscope and calculate the number of viable cells per mL. Unfortunately, due mainly to dye limitations and operator variability, this method is incredibly prone to error. Automated cell counters provide a more robust approach since they remove subjectivity from the cell counting process and allow for the adoption of standardized methods. This translates to improved reproducibility, while also speeding up workflows.
Antigen presenting cells dissected from a mouse spleen are analyzed for viability and count on the CellDrop.
One of the main limitations of Trypan Blue is its inability to resolve nucleated cells from debris, which can lead to counts and viability being overestimated, particularly in the case of more complex samples. Although immortalized cells that have been trypsinized from a flask usually have little associated debris, primary cell samples contain significant quantities of debris and non-nucleated cells, making accurate counting with Trypan Blue extremely difficult. A further disadvantage of Trypan Blue is that it can be toxic if used incorrectly, causing cell death during counting. It may also be incorporated into live cells during extended incubations, thereby skewing results.
Fluorescence-based counting overcomes these problems by using specialized reagents to bind nucleic acids, leaving cellular debris and non-nucleated cells unstained. Acridine Orange (AO) / Propidium Iodide (PI) is the recommended method, whereby AO stains the nucleic acids of all cells in a sample and PI stains only the nucleic acids of dead cells. AO/PI staining is ideal for counting primary cells—even clumpy cell populations such as primary hepatocytes—as well as whole blood samples, other samples containing significant debris, and irregular or small cell types.
While automated systems take a lot of the hard work out of cell counting, it is important to follow best practices for obtaining the highest quality images and most accurate data. This begins with correctly preparing cells prior to loading to ensure results are representative of the stock sample. For immortalized cell lines, this includes trypsinizing to completion to minimize clumps and mixing immediately before loading to ensure sample homogeneity. Primary cells typically require gentler handling, for example using an enzyme-free detachment solution and careful resuspension with a pipette for anchorage-dependent samples.
Once cells have been loaded into the cell counter according to the manufacturer’s guidelines, it is important to check the focus. This should be adjusted on live cells in the brightfield channel until the cell membranes appear as a dark ring around a bright white center. Next, a suitable exposure should be selected. For fluorescence exposure, it is important to maximize the intensity without overexposing, which would allow the fluorescent signal to exceed the cell boundary.
In addition to improving cell counting consistency, automated systems can save researchers considerable time by streamlining workflows. Immuno-oncology is just one of many research fields benefiting from these advantages, having, for example, leveraged the capacity of automated cell counters to rapidly quantify live and dead cells in both immune tissues and tumors while identifying morphological differences in these mixed cell populations using size gating.
Within a recent study to investigate how Ly-6 proteins regulate the activity of helper T cells in an oncology setting, researchers transplanted tumors into wildtype and Ly-6 knockout mice before measuring the extent of T cell infiltration. Because Ly-6 acts as an inhibitor to helper T cells, it represents a potential drug target to boost the immune response against cancer. Using a DeNovix CellDrop Automated Cell Counter, it was proven possible to determine the T cell/tumor cell ratio more efficiently than using conventional flow cytometry-based methods.
The DeNovix CellDrop™ is an automated cell counter that allows researchers to Count Cells Without Slides™. The instrument provides fast, accurate measurements for a wide range of cell types. To learn more, visit denovix.com.