Reprinted from American Biotechnology Laboratory May 2003
BY COSTAS IOANNIDES
The skill of culturing mammalian cells successfully requires consistent, accurate measurements of cell growth and viability. Cell counts must be taken daily, a task that is not only tedious and time consuming, but also prone to error because of operator subjectivity. To that end, a simple device has been developed that uses well-documented fluorescence microscopy techniques to eliminate operator variances. The automated system detects fluorescent signals bound to cell nuclei, providing accurate cell counts in less than 60 sec. This report details the technology behind the NucleoCounter (New Brunswick Scientific Co., Inc., Edison, NJ; figure 1) and its operation, including data from a run of Chinese hamster ovary (CHO) cells. Alternate counting methodologies, both manual and automated, are also discussed.
Today, most laboratories count cultured cells manually, using a hemocytometer under a microscope to determine total cell number using the Trypan Blue exclusion method to determine cell viability. Because the Trypan blue dye only enters the walls of impaired plasma membranes and is excluded from viable cells, both stained and nonstained cells can be visualized and counted in a light microscope. One drawback, however, is the propensity for staining artifacts, which can lead to higher counts. A second drawback is the proportional increase of stained cells as the length of incubation time between sampling and analysis increases. Furthermore, the naked eye can only differentiate pigment intensities in a limited concentration range of the stain in the hemocytometer chamber. Combining these drawbacks with the potential for cell aggregation, it is easy to see why variation in cell counting is commonplace using this standard methodology. Moreover, it is a common occurrence to acquire cell counts from various cell-counting s