Cell counting is fundamental to in vitro cell culture since it allows for monitoring of cell health and proliferation rates. It is also an essential step in seeding cells for downstream experiments, with accurate counting critical to ensuring experimental reproducibility. Although researchers have for years relied on traditional glass hemocytometers to generate cell counts, there has been a steadily growing trend toward the use of alternative methods. These aim to eliminate user-to-user variance, speed up the counting process, and deliver more reliable results.
Stuart Wood, research scientist at Bactevo, which is developing drugs to treat diseases that involve defects in mitochondrial function, routinely performs assays using HepG2, HeLa, HEK293, and other standard cell lines, in addition to iPSCs. He currently uses disposable hemocytometers to count his cells. “Disposable systems provide several advantages over glass hemocytometers,” explains Wood. “Not only can glass hemocytometers be subject to contamination, but it is also possible for the cover slide to lift slightly if greater than the recommended volume of liquid is introduced to the counting chamber. This can result in counting inaccuracies, which will impact on experimental outcomes. Glass hemocytometers can also be easily damaged.”
Although Wood does not routinely count cells for passaging, he explains that “counting helps those who are newcomers to cell culture learn how to split cells at an appropriate time and at the correct ratio. It’s also extremely useful for more temperamental cell lines, which perhaps can’t tolerate being split too heavily. Furthermore, counting has huge utility when you wish to accomplish multiple transfections with different cell lines simultaneously since it allows you to ensure that all the cells are ready at the same time.”
Wood is a strong advocate of automated cell counters. “Automated systems often provide a live:dead ratio. This can flag up any issues, and is particularly useful when you’re generating a new cell line or performing a transfection as it provides an indication of whether the modification you have made is toxic.”
Automated instrumentation
Beckman Coulter is a name synonymous with cell counting. According to Stephen Munt, cell analysis specialist at Beckman Coulter, the company’s current flagship Coulter Counter is the MULTISIZER 4e. This affords sampling rates of up to 10,000 counts per second, and can detect particles from 0.2 µm–1600 µm in diameter. “We also offer the popular Vi-CELLXR cell count and viability analyzer,” explains Munt. “This instrument automates the trypan blue cell viability test, and provides cell count, cell concentration, cell viability, cell size, and cell image information in a single automated analysis.”
Biocompare’s Cell Counting Search Tool
Find, compare and review cell counters
from different suppliers Search
Jean Qiu founded Nexcelom Bioscience in 2003 to address the issues faced by researchers when counting manually. “At the time Nexcelom was formed, researchers were predominantly using glass hemocytometers to count their cells. We developed a disposable hemocytometer, the CP2, with an all-in-one design to eradicate the need for cleaning or assembly of parts, yet although this was hugely popular it still required that a manual cell count be performed. Extensive customer feedback informed us that not only did researchers not want the hassle of preparing a hemocytometer, but they would also prefer not to spend time at the microscope performing multiple cell counts by eye. As a result we invented our Cellometer Auto T4, a chip-based instrument for measuring cell concentration and viability.” The use of a slide avoids the need for a liquid-handling system, meaning that there is no need for a regular cleaning routine to be implemented, while the included software enhances workflow efficiency by performing calculations for the end user.
Following the success of the Cellometer Auto T4, Nexcelom expanded its product range to include instrumentation for more advanced cellular analysis. “All cells are different,” explains Qiu, “and choosing an appropriate cell counter is not a trivial matter. Although any cell counter can produce a numerical readout, we believe that a truly accurate cell count is purpose dependent, and we’ve designed our next-generation systems with this in mind.”
Tiffany Sidwell, clinical laboratory scientist apprentice at UCLA molecular diagnostics laboratories, is a regular user of Nexcelom’s Auto 2000 instrument, which is specifically optimized for analysis of primary cells from complex heterogeneous samples such as peripheral blood, cord blood, or bone marrow. The instrument uses dual-color fluorescence for identifying both live and dead stained nucleated cells, enabling the generation of accurate viability results even in the presence of debris, platelets, and red blood cells. “We routinely count PBMCs and cryopreserve them at a specific number per vial,” says Sidwell, “and since adding the Auto 2000 to the lab, our samples have become more reliable in terms of viability and accuracy of counts.”
Image: Due to hepatocytes' variable morphology, fragile nature, and tendency to clump, traditional manual counting methods can be time-consuming and inaccurate. Cellometer K2 or Cellometer Vision capture and automatically count bright field and fluorescent images. While the bright field image shows cell morphology the fluorescent image shows live cells stained with acridine orange (green) and dead cells stained with propidium iodide (red). Image courtesy of Nexcelom Biosciences.
For researchers requiring a more portable approach to automated cell counting, MilliporeSigma developed the Scepter™ 2.0 instrument. This handheld device provides similar functionality to many benchtop automated cell counters while enabling counts to be performed within the culture hood. According to Karen Tiano from MilliporeSigma, “Scepter™ 2.0 instrument is easy to use and generates accurate, reliable cell quantitation significantly faster than other methods. For example, a sample at a concentration of 500,000 cells/mL can be counted on average in just 14 seconds when using a 60 micron sensor; a manual count with a glass hemocytometer would take about 10 times longer. And our Scepter 2.0 instrument requires just 100 µL of diluted cell suspension per count, which is helpful when volume is limited.”
The Scepter 2.0 instrument reportedly produces more accurate and precise counting of low concentration samples compared with a hemocytometer. Data analysis can be performed on the device, or alternatively results can be uploaded to a computer. High-resolution measurements of cell size and volume are used to generate a histogram distribution. “Gating allows you to focus on your population of interest, while quantitative data on cell populations can be used to evaluate the quality and health of the culture,” Tiano adds.
While manual counting has not yet fallen from favor, companies such as Beckman Coulter, Nexcelom Bioscience, and MilliporeSigma are doing all they can to provide researchers with affordable measures to improve the efficiency and accuracy of their cell counts. Whether a disposable hemocytometer, an automated benchtop system, or a handheld device is chosen, these modern systems supply huge benefits and their utility is flourishing.