Counting Stem Cells with the Countess® Cell Counter

Optimizing Countess® Settings
The Countess® Automated Cell Counter is equipped with programmable settings that allow the user to adjust analysis parameters—sensitivity, maximum and minimum cell size, and circularity—in order to tailor a protocol for a specific cell type. Post-count data manipulation can be performed on the instrument, or using the Countess® software with archived data. To measure the same cells with different sensitivity and circularity settings, separate readings must be taken. In contrast, the size gate can be set before or after cell counting.

To determine the optimal settings for counting stem cells using the Countess® Automated Cell Counter, human embryonic stem cells (hESCs) were cultured using a variety of methods: using a feeder cell layer in a culture dish; using Geltrex™ matrix– coated culture dishes; and using StemPro® feeder-free media. The cells were then counted on the Countess® Automated Cell Counter and compared to the counts obtained on a hemocytometer. GIBCO® rat fetal neural and mouse embryonic stem cells, as well as StemPro® human adipose-derived stem cells were also counted using both techniques. We found that all of these stem cell lines cultured using feeder-free methods did not require further parameter modifications from default settings to obtain accurate cell concentrations (data not shown) from single-cell suspensions; however, the hESC cells cultured using a feeder cell layer needed some adjustments to the analysis parameters for more accurate cell counting.

Adjusting Countess® Parameters
Stem cells grown in some conditions, such as on feeder layers, often exhibit variable morphology, including lighter, less-defined edges; this may make it difficult for the cell counter to distinguish which cells to include in the count (Figure 1A). To alleviate this issue, you can simply increase the sensitivity setting, allowing the instrument to include these cells in the count (Figure 1B). By increasing the sensitivity setting, however, the instrument is also more likely to count debris that was previously ignored. Because stem cell debris is often smaller than the size of the cells to be counted, increasing the minimum cell size parameter will counter this problem by excluding the debris from the count (Figure 1C).

Our suggested parameters for stem cells grown on a feeder layer are listed in Table 1. Using these parameters, we were able to successfully count and determine the viability of hESCs grown on fibroblasts with a high degree of accuracy, and over a broad range of cell concentrations (Figure 2, Figure 3).

Table 1. Recommended Countess® parameters for stem cells grown on a feeder layer.

Parameter	Default settings	Suggested stem cell settings
Sensitivity	5	8
Minimum cell size	5	8
Maximum cell size	60	60
Circularity	80	80

Summary
Automated cell counting bypasses tedious manual operations and calculations required with the standard hemocytometer, while offering generally greater accuracy and precision. The Countess® Automated Cell Counter also makes it practical to count multiple replicates and to design experiments with large numbers of samples for counting, even with stem cells.