Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Arduous though it can be, accurate cell counts are vital for successful experiments. Cell biologists count cells to determine concentrations for plating in culture. They count live and dead cells to assess viability.
The oldest way to count cells, still used today, is the most straightforward: Put the cells under a microscope and count a defined volume using a hemocytometer. Some have automated that method in automated image analysis, while other methods rely on light absorbance (spectrophotometers), electrical resistance (Coulter counters) and marker abundance (flow cytometers). The method you choose may depend partly on cell type and experimental aims, not to mention available instrumentation, but all will give you the same answer. And all can benefit from some fundamental cell-counting tips.
Sample preparation
Several factors will help you get the most accurate count possible. “A good cell suspension for counting is a representative cell suspension of the original cell stock,” says Cindy Neeley, field application specialist at Thermo Fisher Scientific.
Ideally, counting samples contain only single cells, but often they also contain debris and cell clusters. Debris usually can be removed by filtering, but cell clusters are a stickier problem. “Good sample prep is key,” says Veronika Kortisova, product manager for the Gene Expression division at Bio-Rad Laboratories. “Samples should be trypsinized to completion to break cell clusters apart.” Gently pipetting the sample up and down a few times can aid in further dissociating clusters if needed. (An image-based automated cell counter, like a microscrope and hemocytometer, provides immediate visual feedback on sample quality, she adds, something other counter designs do not do.)
Even with enzymatic and mechanical disruption, cell clusters sometimes remain. Adherent cells, especially, tend to form clusters when in contact, so it may help to keep culture vessels on a shaker or rotating platform. Still, says Kortisova, some counters can handle that situation. “Doublets [or two cells stuck together] often remain in the sample, and if an automated cell counter is used for analysis, then it should be able to count individual cells in the cluster,” says Kortisova. Bio-Rad’s TC20™ cell Automated Cell counter Counter can accurately count clusters of up to five cells, she says.
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Another tip for counting samples accurately is to vortex or mix them immediately prior to pipetting, and to pipette from the middle of the tube rather than the top or bottom, where there may be variations in cell concentration as the cells settle, says Kortisova.
Cell concentration
Another key parameter is cell concentration. For most cell types, the optimum range is 1 to 3 million cells per milliliter, says Frank Hsiung, Senior Scientist in the Gene Expression division at Bio-Rad Laboratories. “If the concentration is too low, then the number of cells in the counting field may not be representative of the actual concentration in the stock solution,” he says, using the hemocytometer method as an example. But if the concentration is too high, then cells may begin aggregating or overwhelming the method, leading to counting errors.
Neeley recommends diluting the counting sample so that each large square on the hemocytometer contains five to 100 cells. “Count cells in at least the four large corner squares of the nine large squares on a hemocytometer,” she says. “Within each counting square, always include cells on two of the four borders.” This increases the chance that the counted cells better represent the original sample.
Getting an accurate count
Although many cell-culture practitioners still use hemocytometers, automated, vision-based counters can provide higher throughput for labs that generate large numbers of samples. These systems use brightfield or fluorescent microscopes with CCD cameras to image cells.
"The cell count is determined by applying focusing algorithms onto a visual representation of the cells," says Debra Hoover, technology manager in cell biology and cell-culture systems at EMD Millipore. "One limitation can be the robustness of the focusing algorithm that is used."
With any automated cell counter, preparing a good sample includes following the specific instrument’s guidelines. “Stay within the recommended concentration range of the system or apparatus that you are using for cell counting,” says Matt Rhyner, strategic marketing manager, particle characterization at Beckman Coulter Life Sciences.
Increasingly, automated cell counters are easy-to-use, time-saving devices. For example, ChemoMetec's Nucleocounter® NC-200 is an automated imaging-based system that counts cell nuclei stained with the fluorescent dye, propidium iodide. fluorescence-based imaging system. With minimal hands-on time, researchers can receive a cell count and viability measurement in less than a minute. "The NC-200™ uses a precalibrated cassette to load samples, so the instrument itself and the sample [do] not require a pretreatment step or a calibration step, providing a truly objective reading without any human manipulation," says Warren Ang, product manager for bioprocess at Eppendorf North America, which used to distribute the NC-200.
Flow cytometry counts cells by funneling them one by one through a narrow opening, where they are counted as they pass a laser. Some flow cytometers require comparing your sample counts to known concentrations of standards comprised of beads. Other systems, like Life Technologies’ Attune® Acoustic Focusing Cytometer, don’t require standards, says Mike Olszowy, R&D leader for cell biology at Life Technologies.
Olszowy says cytometers that use bead standards function on the assumption that the instrument treats beads the same as cells, so there is “potential for ... error in this latter approach via pipetting, bead settling, insufficient mixing [and] the time the tube sits on the cytometer.” This possibility need not deter you from using bead standards, but it is a valuable reminder that proper pipetting techniques and well-timed vortexing or mixing are important in cell counting.
Coulter Counter® analyzers are also widely used for cell counting. According to Hoover, they "are the gold standard for clinical cell counting due to their reliability and statistically robust methodology." Coulter Counter analyzers use electrical impedance to measure the volume of particles as they travel, one by one, through an aperture.
Easy as they are to use, Coulter Counter analyzers also are susceptible to cell clumping, a problem called “coincidence,” in which two cells enter the aperture simultaneously, skewing counting results. "When using a Coulter-principle instrument, it is important that the cell-suspension solution contains additives to reduce undesired cell clumping, that the cells are dilute enough to prevent coincidence ... and to select the optimum size aperture for the cell type to be analyzed," says Rhyner.
Rhyner also notes the importance of a counting instrument's dynamic range—particularly if a lab studies cell types of different diameters. One such device is the Multisizer™ 4 Coulter Counter from Beckman Coulter Life Sciences. EMD Millipore also offers a handheld, impedance-based cell counter called the Scepter™ Automated Cell Counter, says Hoover.
Especially small cells (diameter under 10-µm) can pose a counting problem for hemocytometer or imaging-based methods, because smaller cells are more likely to be in different focal planes than larger cells. Kortisova notes that letting the cells settle for about 30 seconds prior to counting can help, but they still may not reside in exactly one focal plane. "This complicates viability analysis, as live cells may look dead when imaged in the wrong focal plane and vice versa," he says. A researcher can refocus the microscope on individuals cells as needed when counting by hemocytometer, but automated cell counters with one focal plane cannot be adjusted this way. If small cells are an issue, automated instruments such as Bio-Rad’s TC20 cell counter use multiple focal planes when analyzing viability.
No matter which method you use for counting cells, you’re in luck. Counting methods have been fundamental cell-culture tools for decades. You should have no problem finding resources, whether online or next door, that can help you work through your problems and move your research forward.
Image: A hemocytometer. By Flickr user “Todd,” uploaded to Wikimedia Commons.