Each procedure may affect cellular growth, skewing measurement of the effects. This is especially true when subject chemicals affect cell proliferation, yet the experiment aims to quantitate a different parameter such as metabolic activity, which then needs to be normalized against cell number.Ĭounting cells is required when adherent mammalian cells are cultivated for numerous other experimental applications such as measuring protein overexpression or RNAi gene silencing. In cell culture experiments where the effect of one or more chemicals on cell number must be assessed, it would be ideal to know precisely how many cells have been plated into a well or a subregion of a well at the beginning of an experiment, how many cells were alive days later before addition of the chemical, and how many cells were present at the end of the experiment.
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This method will allow routine cell counting using a plain bright-field microscope without cell-line modification or cell staining. On average, bright-field images produced the same counts as fluorescence images, with less than 5% error. Counts from day-old and freshly seeded plates were compared in a range of densities, from sparse to densely overgrown.
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The procedure was benchmarked against manual and automated counting of fluorescently labeled cell nuclei.
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Contrast may be enhanced by swelling cell bodies with a brief incubation in PBS. The technique allows robust, automatic cell counting from a single bright-field image in a wide range of focal positions using free, readily available image-analysis tools. Refraction by each cell body generates a sharp, bright spot when the image is defocused. Cells are illuminated through a color filter and a pinhole aperture placed between the condenser and the cell culture surface. We present a very simple procedure yielding high-contrast images of adherent, confluent cells such as human neuroblastoma (SH-EP) cells by ordinary bright-field microscopy.