The routine and accurate counting of live and dead cells in a cell culture is central to maintaining healthy and productive cultures. Whatever the technology used, the basic method involves staining the cells (usually with a nuclei-staining dye) and counting the indicated cells using a hemocytomer, a cell counting chamber invented by the 19th century French anatomist Louis-Charles Malassez. Typically, healthy live cells exclude the dye (such as trypan blue or propidium iodide), while dead ones do not, so counting involves two stages—counting dead cells, then adding detergent that enables dye to enter all cells, and then counting total cells.
In manual counting, researchers count cells by eye through a microscope. In automated counting, a computer connected to a camera and microscope counts the cells. Even while automated cell counting technology flourishes, there are some instances where one really does want to rely on the old-fashioned manual method—looking through the microscope and counting by eye. Here’s a look at when to use which method, and some new tools available.
Why use manual cell counting?
One of the most common reasons to use manual cell counting is to rely on human judgment to count cells—for example, if expert discretion is needed to discriminate between cells types in a mixed sample. As such, manual cell counting is in some ways the gold standard of cell counting. Another reason may be simply that only occasional counts are needed, so using an automated system might be overkill. “Labs still do it,” says Mike Mortillaro, owner of Bulldog Bio. “The human eye can better discriminate between live and dead cells, and there are also different types of established, validated protocols out there for cell counting that labs like to follow closely.”
Bulldog Bio offers disposable hemocytometers to make manual counting easier. Made of plastic, they are less fragile than traditional glass hemocytometers, contain pre-etched counting grids, and an integrated cover slip so that no cover slip is needed—you simply pipette 10 µL of cell sample to the port of the counting chamber. “Our disposable hemocytometers are especially great when you are working at biosafety level 2 or higher situation, where you have to be concerned about contamination,” says Mortillaro.
Advantages of automated cell counting
Despite the advantages of human discretion in manual cell counting, human involvement can also lend disadvantages. The advantages of automated cell counting include fewer sources of human error, more repeatable results, faster counting, and more variables measured. An array of automated counting systems are available today, running the gamut from straightforward and minimally automated, to sophisticated cell analysis with complete automation.
Bulldog Bio offers an example of the former in their ADAM MC Auto Cell Counter, which combines a microscope with fluorescence detection of propidium iodide nuclei staining and analysis. “The fluorescence detection results in low variability, approaching that of a flow cytomer, but not as expensive and easier to work with,” explains Mortillaro. It uses a chip nearly identical to the hemocytometers for manual counting, but lacking the etched counting grid. “It reads 22 times across the entire chip and then averages the reads, which lowers variability,” he adds.
Another easy-to-use system is MilliporeSigma’s Scepter™ 2.0 Handheld Automated Cell Counter, which is the only handheld cell counter available today. “The system employs the Coulter principle to deliver accurate cell counting in less than 30 seconds, and displays information regarding cell concentration, average cell size, average cell volume, as well as a histogram of size or volume distribution,” according to Michael Omartian, director of product marketing of cell culture workflow tools at MilliporeSigma.
DeNovix offers a new automated cell counter, the CellDrop, that operates without slides. Instead, it uses optical-grade sapphire surfaces that wipe clean between uses. Using their patent-pending DirectPipette™ technology, the CellDrop creates a variable height sample chamber (controlled by their EasyAppsⓇ software), allowing a wide range of cell densities. For example, standard is a 100 µm chamber with 10 µL cell suspension. But “for high density samples, a lower chamber height and smaller volume is used, avoiding the need for dilutions,” says Andrew Jones, market development manager at DeNovix. “For low densities, the chamber height can be raised to 400 µm, allowing a greater sample volume and more accurate measurements. This avoids the need for time-consuming centrifugation steps to concentrate samples.”
The CellDrop BR analyzes brightfield images, and the CellDrop FL analyzes dual-channel fluorescence plus brightfield images. Pre-configured apps for common counting and viability assays are available, such as trypan blue, acridine orange, propidium iodide, and GFP expression assays; custom apps and protocols can also be created. “For applications that require the cells to be contained when reading due to potential biohazard, the CellDrop also has the option to use disposable or reusable slides without the need for additional adaptors,” notes Jones.
Many applications today require not only cell counting, but also monitoring of key cell health indicators. Nova Biomedical’s BioProfile® FLEX2 cell analyzer gives automated cell counts in addition to other important culture parameters, including pH, osmolality, gases, and other chemistries. Its ability to return 16 tests from one sample reduces errors from manual pipetting samples into multiple analyzers for multiple tests. The chemistry and gas biosensors are integrated into a MicroSensor Card™ the size of a credit card. The FLEX2 uses the common Trypan blue dye exclusion method for cell counting, with a unique twist. “We use a moving flow cell coupled with a high-resolution camera and optics to enable better differentiation of the cells from the background,” says Matt McRae, product manager at Nova Biomedical. “The FLEX2 is unique in the cell analyzer market, being the only one that incorporates cell counts with the other parameters.”
Nova Biomedical’s analyzers are fully automated with robotic systems, including sample preparation. “The only thing you need to do is take a sample from your cell culture and position it in front of the analyzer,” says McRae. “The analyzer does all the dilutions, mixing with trypan blue, and cell counting.” While many cell counters require an initial dilution step, Nova Biomedical’s system “can handle cell counts from 100,000 cells to 80 million cells per mL without any external dilutions,” he says. Removing the need for a pre-dilution step can prevent another source of human error.
Whether you need to count cells occasionally or routinely, the chances are good that you will find a configuration to meet your needs in the array of manual, handheld, robotic, and cell analyzer systems available today.
Preparing cell samples for accurate counting
One of the most important factors in improving cell counting accuracy is consistency with sample preparation. “The most common pitfalls with cell counting are inherent variation and user subjectivity or error,” says MilliporeSigma’s Omartian. “Consistency in sample preparation through implementation of proper tools and protocols will help to ensure accurate and consistent cell counts between experiments.” He adds that automated tools should also be routinely calibrated for the best performance and accuracy.
Another important factor in sample preparation is “proper mixing of the cell culture prior to withdrawal of the sample for counting,” explains Nova Biomedical’s McRae. “It’s important to make sure that the sample is homogeneous before presenting it to the analyzer.”