Quantitating cell number is more accurate using the Quantos cell proliferation assay kit
Leslie S. Arrington
Genetic Applications LLC
The Quantos cell proliferation assay kit provides a simple, reproducible method to assess the number of cells present in various tissue culture dish formats. In this study, we compared how well the kit performed compared to another commercial fluorescence-based cell proliferation assay product. The data demonstrated that the kit more accurately assessed cell number in the range of cell number frequently found in commonly used culture dish formats.
Researchers are frequently interested in determining the effects of various drugs, toxins, or experimental conditions on cell proliferation. With Stratagenes new Quantos cell proliferation assay kit, it is easy to detect cell number directly in the tissue culture well: After aspirating cell culture media, add the Quantos dye reagent, and measure fluorescence in each well using a fluorometer. Since the Quantos dye fluoresces only when bound to DNA, the assay is insensitive to changes in cellular RNA concentrations or proteins. Relative fluorescence units from the sample wells are compared to a standard curve prepared beforehand. In this way, the number of cells in each well of a tissue culture plate can be quickly and accurately determined.
We set out to determine how well the Quantos cell proliferation assay kit compares to another widely available cell proliferation assay product that uses a proprietary green fluorescent dye. Standard curves were made using adherent and suspension cells at various cell densities ranging from 0 to 80,000 cells/well in a 96-well format. The results were plotted, and trendlines for each assay were calculated.
Results for an adherent cell line are plotted in Figure 1. COS-1 cells were counted using a hemocytometer, then plated at the densities shown in the graph. Cells were allowed to attach over four hours, then quantitated according to the manufacturers instructions for each kit. The resulting graphs showed that the Quantos cell proliferation assay kit provides a linear relationship of cell number versus fluorescence units (r2= 0.980). However, the other assay demonstrated a less linear relationship (r2= 0.897).
Results for a suspension cell culture are plotted in Figure 2. BJAB cells were counted using a hemocytometer, then plated at the densities shown in the graph. Cells were then quantitated according to the manufacturers instructions for each kit. Once again, the graph indicated that using the Quantos cell proliferation assay kit elicits a better linear relationship of cell number versus fluorescence units (r2= 0.974), compared to the other assay (r2= 0.938).
In both the adherent and suspension cell lines, the other assay demonstrated a plateau over 50,000 cells. In fact, the manufacturers product information sheet states the following: The linear range of the assay under these conditions is from 50 to 50,000 cells per 200 l sample. Since the number of cells in each tissue culture is determined by extrapolation from a standard curve, this nonlinearity makes calculating the cell number above 50,000 cells difficult and inaccurate.
To determine whether the observed competitors plateau of 50,000 cells could cause errors in quantitating cell number in a typical cell sample, we plated CHO cells at 100% confluency in 96-well culture dishes. Since CHO cells are adherent cells and typically form a monolayer, this test demonstrates the minimum number of cells that will cover the well floor in a 96-well culture dish. As Table 1 shows, even these cells will exceed 50,000 in cell culture dish formats larger than 96-well. Many other common cell lines, such as COS-1, form multiple layers of cells and grow to over 100,000 cells per well in 96-well culture dishes (data not shown). Additionally, suspension cell types typically grow to densities of 1,000,000 cells/ml, resulting in 200,000 cells per 200 l sample.
Monolayer Adherent Cells at Approximate 100% Confluency
Number of Cells in Various Dish Formats
The number of CHO cells required for 100% confluency in 96-well format culture dishes were determined by adding serial dilutions, allowing cells to attach over four hours, then rinsing and quantifying cell number. The number of attached cells plateau at 50,000 cells, regardless of how many additional cells are added; this indicates the approximate maximum number of monolayer cells that can attach on a 96-well format. The number of cells required for approximate 100% confluency in 48-well and 24-well formats were estimated using a relative well-floor surface area.
Therefore, suspension cell cultures can significantly exceed the 50,000 cells per well in 96-well culture dishes. These findings suggest that the number of cells per well can easily exceed 50,000 in many cell lines, even in 96-well culture dishes.
To quickly and accurately measure cells at numbers that can exceed 50,000 cells per well, the Quantos cell proliferation assay kit is superior to other commercially available products. The kit accurately quantitates cell number by providing a linear relationship of cell number versus fluorescence units, in the cell range typically found in common tissue culture well formats; thus, counting cell number is more accurate.