The Next Generation in Cell Counting
Optimizing Countess® Instrument Settings for Stem 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® hESC SFM feeder-free medium. 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 setting 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 Parameters for Stem Cells Grown on a Feeder Layer
Our suggested settings for stem cells grown on a feeder layer are shown in Table 1. Using these settings, 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 1. Adjusting Countess® instrument parameter settings for stem cells grown on a feeder layer. hESCs grown on a mouse fibroblast feeder layer were dissociated into a single-cell suspension using TrypLE™ Express dissociation enzyme and counted using the default settings on the Countess® Automated Cell Counter. Blue circles outline cells that are counted as viable; red circles outline cells that are counted as dead; and black circles indicate objects that are excluded from the count due to the size settings. (A) Some of the less-developed and less-defined cells are not included in the count, as indicated by the cells that are not marked with a blue, red, or black circle. (B) A different slide of the same single-cell suspension was counted after increasing the sensitivity setting from 5 to 8, resulting in fewer cells excluded. (C) The image in (B) was recounted, leaving the sensitivity setting at 8 while increasing the minimum cell size from 5 to 8. As shown, smaller feeder cells and debris were excluded from the count.
|Parameter || Default Settings ||Suggested Stem Cell Settings |
|Sensitivity || |
|Minimum cell size || |
|Maximum cell size|| |
|Circularity || |
Figure 2. High accuracy of hESC cell counts (Left panel) and viability (Right panel). hESCs were concentrated, serially diluted 3:1, and counted using the Countess® Automated Cell Counter and the C-Chip disposable hemocytometer (Digital Bio). Data points represent 3 independent measurements on the Countess® instrument or hemocytometer. Viability was determined by trypan blue exclusion.
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