Kathrin Renner-Sattler’s lab went through hell in 2015. The reason? The lab’s automated cell counter broke down after a decade of use, forcing the scientists at the University Hospital of Regensburg in Germany to count manually, using a hemocytometer.
Normally the group, which studies tumor-infiltrating immune cells, relies on a CASY cell counter from OMNI Life Science. For Renner-Sattler, automation offers three clear advantages. First, it standardizes the process of cell counting across different lab members, who might count differently by eye. Second, it enables them to get through up to 100 samples on their busiest days, painlessly; some counters now require just seconds to tally a culture. Third, the machine offers more than just a number. Like other automated counters, it also gives the distribution of different cell sizes, a good indicator for the health of the cultures. “You can easily see whether your cells are all right or not,” says Renner-Sattler.
About 50% of large cell-culture labs have switched to automated counters, estimates Michelle Rule, senior global product manager at MilliporeSigma. Cell counts are a key element of quality control, considering that the density of cells in an experiment could change their behavior or gene-expression patterns, notes Siegfried Sasshofer, director of marketing detection at TECAN. Automated counters tend to offer more accurate counts than a scientist staring down a microscope at the gridded pattern on a hemocytometer. Sasshofer thinks they’re worth it for scientists who count more than five to 10 cultures daily, or for those who want to ensure cultures are always counted the same.
Cell counters are most popular in labs working with animal cell lines or primary cells, but counters can also be of use for scientists working with yeast, protozoa and algae, says Joachim Pavel, business director at OMNI Life Science. He finds that lately, microbiologists who work with bacteria are interested in the CASY, as well. The machine offers a census right away, as opposed to plating cultures and waiting a day or two to count colony-forming units.
Image-based counters
There are two main types of counters used by researchers: those based on microscope images and those based on the passage of cells through a pore, as in flow cytometry.
Flow cytometers themselves can also quantify cells, of course, though it’s not worth purchasing one just for counting, advises Ana Kim, the U.S. regional manager for Logos Biosystems.
Image-based counters work much like manual counting; they just take the place of a human scientist’s eyes and counter. They work quickly but typically only count as many cells as there are in a single field of view. For example, for the Countess II instrument from Thermo Fisher Scientific, that might range from 10 or 20 cells up to a little more than 1,000. If there are relatively few cells, the machine extrapolates from a small, counted number and might not be very accurate. One solution is to perform two counts and average the results, suggests Sadaf Atarod, a post-doc at the Boston University School of Medicine Center for Regenerative Medicine. Some imagers also let users request multiple pictures, boosting accuracy.
One key feature to look for in an image-based counter is an autofocus option. If the image isn’t in focus, it won’t be counted or recorded correctly. Another consideration in selecting an instrument is how well the counter deals with aggregates. Many counters have algorithms to differentiate cells in clumps but typically only for flat, 2D aggregates, says Stephen Full, a product manager at Thermo Fisher. Counter makers are still struggling to identify individual cells in 3D cell balls. Pipetting or vortexing can also help resolve aggregates before counting, suggests Bodo Ortmann, a principal investigator at Lonza.
In addition to counting cells, counters can differentiate between live and dead cells using a vital stain such as Trypan blue, which only traverses the membrane in dead cells and stains blue. Some counters go beyond brightfield imaging and include fluorescence imaging, too. That enables scientists to detect signals beyond viability, such as GFP, which is often used to monitor transfection efficiency.
Coulter counters
Counters that tally cells moving through a pore are based on the Coulter principle.
They contain two compartments, each filled with electrolyte and separated by a pore. An electrical current crosses the pore. Every time a cell gets in the way, it provides resistance and alters the current. The machine detects and counts these interference events.
Because of the microfluidic systems involved, Coulter systems tend to be more expensive than their image-based counterparts. Also, they tend to run a bit slower than systems that use imaging. However, they usually count more cells. The CASY, for example, can count up to 20,000 animal cells or 200,000 bacterial cells from a single one-ml sample, leading to high accuracy.
Coulter-based systems can also detect aggregates, simply by determining the volume of the particle passing through the pore, Pavel says. Because the machine monitors the volume of the typical cell in a solution, it can extrapolate how many cells are in a big glob of them. However, Renner-Sattler still prefers to count by eye with very sticky cell types, such as pancreatic cells.
Coulter counters can determine cell viability, Pavel says, because one of the first things that happens to a dying cell is its plasma membrane develops holes. After that happens, the machine’s current can go through the cytoplasm, but it is still slowed by the intact membranes of the nucleus and surrounding endoplasmic reticulum. The counter detects this as a smaller structure.
One more important factor to consider when making a selection of either type of cell-counting system is that the cell solution count falls within the machine’s preferred range; otherwise, the results won’t be accurate.
If necessary, one can dilute suspensions with too many cells or centrifuge suspensions with too few cells to concentrate. It’s best to avoid centrifuging, if possible, advises Ortmann. It can stress the cells. For that reason, he prefers counter systems that don’t require scientists to resuspend cells in a buffer other than their usual growth media.
Choices
Accuracy is the key consideration when choosing a counter, Atarod says. Cost is also important, not only for the counter but also for the consumables—such as disposable slides—needed for each sample. Some companies have begun to offer washable, reusable slides to minimize ongoing costs. Prices vary, depending on features; a counter can cost a few thousand or even tens of thousands of dollars. It’s worth paying for a quality machine, Renner-Sattler advises.
Scientists should also look for a simple user interface. And bench space may be a consideration; although most counters are benchtop machines, MillporeSigma’s Scepter is a handheld device, much like a pipettor. “It can easily be taken to your point of research,” says Rule.
In addition to the companies mentioned above, other manufacturers of automated counters include Bio-Rad, Beckman Coulter, Bulldog Bio, ChemoMetec, Molecular Devices, Nexcelcom and Olympus. Researchers have many options when selecting a cell counter.
Whether monitoring cell growth or performing detailed cell-based assays, it is important to understand the health and number of cells you are working with, as these are two important factors that will influence your result.
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