A novel high throughput approach for quantification of cell density
Negmadjanov U, Holmuhadmedov E, Godic Z, Mirza M, Emelyanova L. A novel high throughput approach for quantification of cell density. J Patient-Centered Res Rev. 2014;1:150.
Presented at 2014 Aurora Scientific Day, Milwaukee, WI
Background: Current approach to cell counting using hemacytometer is limited by requirement for high cell concentration and is prone to error. In biological experiments using cells from human cardiac tissues with limited number of cells, this approach results in large variation in cell counts. Here, we demonstrate the utility of a novel approach using a 96-well microplate that accurately provides the density of cells as low as 15,000 cells/cm2, which fulfills an unmet need in experiments with limited cell availability.
Purpose: To develop and test the accuracy of a high-throughput 96-well microplate assay in assessing the cell density in comparison to existing methods. Methods: NIH/3T3 fibroblasts were cultured and differentiated and grown to different cell density. Cell number obtained using hemacytometer was compared to the total fluorescence of propidium iodide, binding to the nuclei of cells permeabilized with Triton X-100 (0.25%), and assessed using multiplate reader. In addition, the total activity of lactate dehydrogenase, an intracellular enzyme, was used to assess the total volume of cytoplasm released from permeabilized cells. Furthermore, the ratio of live/ dead cells was determined by propidium iodide-positive cells and lactate dehydrogenase activity before and after permeabilization in each well of the 96-well plate.
Results:There was a linear relationship between increasing intensity of propidium iodide fluorescence with the density of the cells in the 96-well microplate (ranging from 5,000 to 100,000 cells/cm2). Similarly, linear relationship was observed between the intensity of propidium iodide fluorescence and cellular lactate dehydrogenase activity in corresponding wells. At low cell density (
Conclusion: Proposed propidium iodide and lactate dehydrogenase assays are useful tools for quantification of cell number in high-throughput manner with greater accuracy at low cell density, higher reproducibility and overall time saving. This assay is especially useful in experiments using limited cell number such as cells isolated from the human heart.