by Caitlin Smith
Counting cells by eye, with a hemocytometer and a microscope, may be just perfect for some applications. But for many others, especially those that require counting large numbers of cells, automated cell counting is simply more efficient and more accurate. Automated cell counters come with a variety of counting technologies and attributes, but all of them bring to a lab the benefits of removing operator subjectivity and significant time savings. “Cell counting may be a routine lab procedure, but the accuracy of a cell count can make or break an experiment,” says Grace Johnston, product manager at EMD Millipore. “Incorrect cell counts force you to repeat experiments, wasting your time and reagents.”
Other factors to consider when looking for an automated cell counter include which cells you will be using, your level of required throughput and how much lab space you can devote to the counting device. Wenlan Hu, senior market development manager at Life Technologies, also notes the importance of asking: “What cell information do you need and at what complexity? How accurate do the counting results need to be for your downstream experiments?” Most automated cell counters are based on dyes (including an indicator used to measure cell viability), flow cytometry and/or electrical impedance.
To dye or not to dye?
Using a dye that stains nuclei—for example, propidium iodide (PI)—is a common method to mark cells for counting. Trypan blue also can be used for viability analyses, as it is actively excluded from live cells but stains dead cells.
Bio-Rad recently launched its TC10 automated cell counter, which uses auto-focusing to eliminate manual focusing by the researcher. “This is especially important when assessing cell viability—a small difference in focus can have a dramatic impact on results,” says Veronika Kortisova, cell systems product manager at Bio-Rad Laboratories. “Light scattering that causes some live cells to appear dead can also impact viability analysis. The TC10 uses a multifocal plane analysis for viability. Every cell is analyzed on multiple focal planes to provide accurate results.” The TC10 system is used primarily for counting cultured mammalian cells, but it is also used for smaller organisms (e.g., plankton, spores and amebas). Researchers using the Bio-Rad Bio-Plex system also use the TC10 device for counting Bio-Plex beads.
Kortisova adds that the TC10 counting slide consumables are designed for greater accuracy. “The TC10 counting slides are unique because they ensure even sample distribution regardless of [the] user’s pipetting style, leading to accurate and reproducible results,” she says. “The sample distribution in the slide is very important for the final result. Researchers can inspect the evenness of sample distribution under a microscope; if they observe spaces without sample, there is a chance these may be located in the imaging area and they can impact the count accuracy.”
Life Technologies Countess® Automated Cell Counter uses trypan blue staining combined with an image analysis algorithm to give accurate cell and viability counts in just 30 seconds. Hu says that one of the unique things about the Countess cell counter is its gating function, which enables users to distinguish cells in a mixed population based on cell size. “Users can then save a particular set of parameters when counting a mixed population for future counting,” says Hu. “This feature is particularly useful when counting stem cells or primary cells, allowing the user to exclude cell debris and/or feeder cells from the final cell count determination. Whereas in some techniques cell clumps can cause the final cell count to be underestimated or cell size to be overestimated, the Countess Automated® Cell Counter accurately counts and defines clumps of cells up to about five or six cells.”
Nexcelom Bioscience’s Cellometer line of non-flow imaging cell counters can perform standard trypan blue exclusion counting in addition to counting one or two fluorescent probes. “Cell counters also include magnifications and varied counting chamber depth designed to count standard mammalian cells; much larger-sized adipocytes [larger than 250 microns]; and the much smaller yeast, algae and platelets,” says Jean Qiu, chief technology officer and founder of Nexcelom. The Cellometer Vision cell counter has optical modules that can be changed by the researcher and cover the entire visible spectrum. Counting with fluorescent probes can be advantageous in some situations because “samples with large amounts of debris require fluorescent probes to label cells of interest,” says Qiu. She also recommends ensuring that the counter software can do what’s needed. For example, sometimes “advanced algorithms are needed for de-clustering clumpy cells and counting irregularly shaped cells, as with Nexcelom’s Cellometer software.”
Another fast counting device is New Brunswick’s NucleoCounter®, an automated cell counting device for cell cultures that counts in less than one minute. The NucleoCounter system uses disposable cartridges called NucleoCassettes™, which contain the fluorescent dye PI for staining cell nuclei. “Because the NucleoCounter detects signals from the stained nuclei and not the cells, the system operates without regard to cell size or morphology,” says Thomas Uschkureit, senior product manager at New Brunswick Scientific. “It is capable of counting a variety of culture types, including CHO, hybridomas and adipocytes; stem cells; and primary cells—as well as yeast and a lot of other cells—without requiring calibration. The disposable cartridges make the system virtually maintenance-free, which makes counting cells faster and easier. Another advantage of the PI-based counting system compared to particle counters using trypan blue exclusion is that the latter sometimes cannot distinguish between cells and air bubbles or aggregated or agglutinated cell clusters. The smaller sample size required, 75 µL,also helps to conserve sample[s] in experiments using rare cells.”
The recently launched Moxi™ Z mini automated cell counter from ORFLO Technologies is unique in terms of viability assessment. “This is the only instrument that provides an electronic viability index without the use of reagents (like trypan blue), and studies have shown that this Moxi Viability Index (MVI) is analogous to using high-end flow cytometers.” says Manju Sethi, VP of Business Development at ORFLO Technologies. "Moxi Z implements a sophisticated electronic algorithm based on a number of factors including cellular impedance measurements, population-based impedance statistics, proprietary software algorithms, as well as empirical data to deliver highly accurate cell count, size and viability measurements."
Going with the flow? Using flow cytometers
Using a flow cytometer as a cell counter can provide a great deal more information. For example, the Accuri® C6 Flow Cytometer® System is a small, fully digital flow cytometer that uses a unique peristaltic pump-driven, laminar-flow fluidics system. The C6 system can measure sample volume directly and calculate absolute cell counts and concentration for any identified population in a sample. “Flow cytometry provides a rapid method to identify or classify cells, although most cytometers cannot directly provide the concentration or absolute count of cells in a sample,” says Grant Howes, vice president of marketing at Accuri Cytometers. “The unique peristaltic pump fluidics system of the C6 allows for direct measurement of cell concentration, yet also provides standard hydrodynamic focusing flow, which is crucial to accurate and precise flow cytometry fluorescence and light scatter measurements. The C6 offers cell biologists a user-friendly cell counter and flow cytometer, all in one.” Accuri finds the most common applications for the C6 system are immunophenotyping, cell viability, cell counting and fluorescence protein analysis. EMD Millipore also offers a line of benchtop flow cytometers. Their Guava ViaCount is a no-wash, mix-and-read assay for determining absolute total cell counts, cell viability and apoptotic percentages.
Another kind of flow cytometer cell counter is Life Technologies’ Attune® Acoustic Focusing Cytometer, which uses ultrasound waves (greater than 2 MHz, as used in medical imaging) to position cells in a single focused line along the central axis of a flow channel. “It does this without high velocity or high volumetric sheath fluid, which can damage cells,” says Hu. “Use of accurate syringe pumps enables absolute cell counting without the use of beads, minimizing cost and sample prep time for researchers.” An advantage of the Attune cytometer is its high sample rate, which is nearly 10 times faster than that of other cytometers. At up to 1000 µL per minute, the Attune system is particularly advantageous when the sample is very dilute or precious. “By uncoupling alignment from hydrodynamic forces and sheath flow, the Attune cytometer allows rapid detection of rare events,” says Hu. “The instrument is designed to collect up to 20,000,000 events per run, enabling detection of those rarest of rare events. Lastly, data collected on the Attune Acoustic Focusing Cytometer exhibits lower coefficients of variation (% CV) and more distinct cell populations when compared to a high-end conventional flow cytometer. This increased precision takes the ambiguity out of counting results.” The Attune cytometer typically is used for rare event detection, cell cycle analysis, cell proliferation analysis and immunophenotyping.
How portable? Handheld counters
Perhaps it is important to you to bring the counter to the cells instead of bringing the cells to the counter. EMD Millipore’s Scepter Handheld Automated Cell Counter is a portable cell counter that measures cells using electrical impedance signals. It is fast (less than 30 seconds) and accurate, allowing researchers to use it at the culture hood rather than bringing cells to another area for counting. The Scepter handheld automated cell counter is used to count cells and perform quality control checks when passing and re-plating cells. “If you are switching from manual counting to automation, you have to trust the instrument you are using,” says Johnston. “The Scepter cell counter displays the cell size distribution in a histogram, so you can gate [select] the cells you want to count and exclude cell debris, for example. Being able to view the cell size distribution gives you confidence that you’re counting only the cells you want to count.”
ORFLO's Moxi Z portable automated cell counter combines the gold standard Coulter principle with patented thin-film sensor technology to provide cell count, size distribution and viability information for particles in a broad size range (2-34 microns) - all in just 8 seconds. The unit is compact enough to fit in the palm of your hand, hands-free to sit inside a hood, easy to set up, and uses Bluetooth technology. “Many image-based cell counters require subjective focusing or are unable to reliably analyze cell size either because they only look at two dimensions, or because the cells often ‘flatten out’ on a slide and give the appearance of being bigger than they actually are,” says Sethi. “That's why researchers who need accurate counts turn to flow cytometers - and these are often complex to use and much higher priced from a capital investment and ongoing costs standpoint. Moxi Z now offers these researchers an attractive alternative.”
EMD Millipore recently released a new sensor for their Sceptor that enables counting of cells down to 3 µm. The new offering provides a highly accurate and affordable method to assess cells or particles this small. Hopefully this cell counter or something like it will satisfy all of your lab’s counting needs.
The image at the top of this article is ORFLO's cell counter, the Moxi Z™.