Manual cell counting—a researcher looking through a microscope to count cells on a grid etched into a glass hemacytometer—was long the only option. Though a solid method when properly performed, its time-consuming and laborious nature is vulnerable to human error and person-to-person variability. Enter automated cell counters, which augment the important task of counting cells—crucial to obtaining high-quality data—with imaging and automation. This article provides expert, practical advice for improving the accuracy of automated cell counting.
Automated cell counters
Automated cell counters offer an increase in throughput, time gained for researchers, and more accurate and reproducible cell counts with human errors removed. Counting is accomplished by optical (brightfield or fluorescent imaging) or impedance methods. Some models use disposable slides, while others such as the DeNovix CellDropTM, instead use a patented, specially calibrated sample chamber. Logos Biosystems’ new dual fluorescent cell counter, the LUNA-FX7™, includes a feature for inserting and reading single slides for a convenient quick count (in addition to multiple slide formats). The LUNA-FX7™ includes the CountWire™ software system, which is 21 CFR Part 11 compliant to support the development of cell therapies such as CAR-T cells. While most automated cell counters are benchtop instruments, MilliporeSigma’s Scepter™ handheld instrument, equipped with WiFi and bluetooth capabilities, offers a more mobile alternative.
See More Automated Cell Counters
Find Automated Cell Counters
from different suppliers Search
Instead of imaging, the Scepter™ instrument counts cells using electrical impedance-based particle detection. “A key advantage of our Scepter™ 3.0 handheld automated cell counter is its high-throughput capability to count thousands of cells per measurement with maximum precision, [requiring] no sample prep, dedicated reagents, or hazardous dyes,” says Michele Halter, segment lead, cell culture tools at MilliporeSigma. Impedance-based counters return cell number and cell size measurements, but do not distinguish between live and dead cells as do imaging-based methods.
Know your counter and method
Know how to use your counter
It is not unusual for researchers to overlook an important function or setting simply because they know basically how the instrument works—especially for something as straightforward as counting cells. Tim Steppe, application specialist at Logos Biosystems (part of Aligned Genetics), recommends spending a half hour to understand what your counter can and can’t do, an investment that might prevent wasting time later. “We had a customer who was comparing different counting protocols and couldn’t figure out why each produced vastly different results,” he says. “They spent about half a day trying to track this down.” The underlying cause was that the protocols had different values set for their dilution factors.
Importantly, becoming familiar with your instrument will help you to optimize the settings for your particular samples. “Automated cell counters come with a range of settings for optimizing image capture and analysis for specific cell types,” says Dan Schieffer, scientific director at DeNovix. “It is worth understanding the available options to ensure captured images are representative of your cell sample, [as] proper instrument focus and exposure is crucial, especially for live/dead viability counts.”
Use the best method
Use the most effective counting method for your sample, and a cell counter equipped accordingly. The best staining method for counting live and dead cells will depend upon your sample type. For example, standard Trypan blue staining is usually sufficient for cultured cells. “Most image-based counters will have brightfield capabilities for counting Trypan blue stained cells,” says Steppe. “However, for more complex sample types like whole blood, dissociated tissues, or primary or stem cells, it is best to use nucleic acid stains like acridine orange and propidium iodide, which require a dual fluorescent counter.” Schieffer agrees that cell types “high in debris, or most primary cell samples, will be best counted using a fluorescence-based method that will distinguish live and dead cells from non-cellular debris,” such as using acridine orange and propidium iodide.
Sample size, volume, and dilution
Sample size and volume affect counting accuracy, and thus proper dilutions are important. “Samples that are too dense or too sparse will be difficult to count and use as representative of the entire sample,” says Schieffer. “When using fixed-chamber automated or manual cell counting methods, users will be required to dilute or concentrate cell samples to ensure they are within the measurable range.” Counting more cells also improves counting accuracy. “In practical terms, for cell counting, this means increasing the volume or amount of sample material that is counted,” by increasing replicates or using a larger sample volume, says Steppe.
Don’t forget practical matters
Good lab practices go a long way toward improving accuracy with any counting method. Start with a clean surface before loading your sample, and mix cells thoroughly prior to removing a sample for loading into the counting chamber. “Cells are not in a homogeneous solution, and will settle quickly in solution so ensure that samples are well mixed immediately prior to loading,” says Schieffer. “Reproducibility errors often stem from neglecting to do this.” Counting inaccuracies can also stem from cell clumps as a result of incomplete or improper trypsinization.
Regularly validating the performance of your cell counter is also wise. “Validations can be performed by either counting a bead solution of a known concentration or, for more exact validations, counting pre-spotted QC slides,” says Steppe, who recommends validating every 1–3 months. A regular visual inspection of the counting data is also a good idea. “Most automated counters give a visual representation of the sample marking which cells are counted,” says Schieffer. “It is important to check this image, ensure that the counted cells match your expectations, and adjust if necessary.”
Because many automated cell counters have the same basic functions, researchers may get the impression that they are all alike—yet they can differ in performance. “We highly recommend that customers demo several models to ensure that at least one can accurately count the range of samples used in their specific applications,” Steppe says. “We’ve seen more than one lab and even some companies negatively impacted by a hasty choice that was driven more by convenience than performance.” In addition to the tips provided here, trying before buying can help to improve the accuracy of cell counting.