My previous response to the problem of variability of TUNEL assay was restricted to the genuine differences in frequency of DNA strand breaks. However, there are often trivial, technical reasons for irreproducibility. One relates to the fact that some investigators collect only adherent cells that are harvested by trypsinization or EDTA, discarding the medium from flasks. Because most apoptotic cells detach, they are discarded with the medium. The "efficiency" of discarding medium may vary from flask to flask, hence the variability in frequency of apoptotic cells.The correct way, of course, is to pool the cells from medium with the these harvested by trypsinization. Apoptotic cells are very fragile and often completely disintegrate during centrifugations. Hence, their frequency (apoptotic index; AI) may vary depending on number of cell rinses, centrifugal force and time, etc. Also, compared to nonapoptotic cells, apoptotic cells have different propensity to attach to the tubing. The variability in handling the samples (including the absolute number of total cells per sample) is then reflected by differences in AI.
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