For decades, hemocytometry was the gold standard for cell counting. A sample of media or buffer containing cells may or may not be mixed with a dye. A precise volume (usually 10 µL) of this mixture is pipetted into a groove in the hemocytometer, where it fills a chamber whose floor has a grid precisely etched onto its surface. One or more small or large square fractions of the grid are counted, and, depending on which area has been counted, a scaleup calculation is performed to return a concentration of cells per milliliter.
So what’s the problem with using a hemocytometer?
Automated cell counters eliminate steps that are vulnerable to human error at virtually every step:
Scepter™ counting is 7 to 10 times faster than hemocytometry-and also faster than other automated counters.
Figure 1.The time required to perform cell counts using various methods was compared using a sample concentration of 500,000 cells/mL. Scepter™ counting (14 seconds on average, using the 60 μm sensor) is significantly faster than other counting methods. Using the 40 μm sensor, Scepter™ counts are complete within 25 seconds, on average (data not shown).
The Scepter™ 3.0 cell counter uses the Coulter principle of impedance-based particle detection to reliably and accurately count every cell in your sample. Coulter impedance is more precise than hemocytometry and automated vision-based counting, demonstrating smaller average coefficients of variation when compared with these methods. Here’s how it works:
As with other technologies that are based on flow cytometry (where cells pass one-by-one through a fluidic channel past a detector), precise data is enabled by the large sample sizes that visual counters can’t achieve because they rely on a scatter of cells spread onto a single microscopic field. The Scepter™ 3.0 counter returns histogram data from thousands of cells per sample, resulting in more precise counts
Figure 2.A range of CHO cell concentrations were measured using the Scepter™ 3.0 counter equipped with the 60 μm sensor, and compared with the same samples measured using the Coulter Z2 with 100 μm aperture. Measurement of concentration was highly linear across concentrations ranging from 100,000 to 500,000 cells/mL using both methods.
Figure 3.Jurkat cells were measured to test accuracy and reproducibility of cell size measurements using the Scepter™ 3.0 Cell Counter with both 40 μm and 60 μm sensors. Results are compared to the same measurement obtained with a Coulter® Counter Z2™ Instrument equipped with 100 μm aperture. Data are from five measurements per sample.
Some automated counters require cells to be in PBS, which can handicap productivity by requiring a buffer exchange. To be an efficient part of cell culture maintenance and experiment prep, a cell counting method should also be able to accommodate cells where they live: in media, with or without FBS supplementation.
Figure 4.Polystyrene beads of known diameter (8 μm) suspended in various cell culture buffers and reagents were measured using the Scepter™ 3.0 Cell Counter with both 40 μm and 60 μm sensors. Results are compared to the same counts obtained with a Coulter® Counter Z2™ Instrument equipped with 100 μm aperture. Data are from three measurements per sample.
Start with a single-cell suspension, diluted to a total volume of 100 μL (recommended) to a concentration of 10,000 to 500,000 cells/mL in a microcentrifuge tube or plate well.
For a more detailed explanation of the counting process, watch our Scepter™ 3.0 demonstration video.