Dear Flowers, I thought that I had included a summary in my last note, but apparently my pasting was not as good as my cutting. So here is my second try: Shari Biogen Steve Mullen<sfm5ff@missouri.edu>: I have used the "Death Detection Kit" from Roche with consistent results. The instructions are straightforward and simple. Dennis J. Young <djyoung@ucsd.ed>: We've see the same thing. There is enzymatic amplification of signal, so unless you have VERY good techniques, you will see great variation. Zbigniew Darzynkiewicz <DARZYNK@nymc.edu>: Apoptotic cells are strongly labeled in TUNEL assay only when they have a large number of DNA strand breaks. This is the case when DNA fragmentation is very extensive, which occurs when internucleosomal DNA sections are cleaved. However, in some cell types (often of epithelial or fibroblast lineage) or instances DNA fragmentation stops at the initial step i.e. generating 300 - 50 kb DNA sections, and does not progress into intenucleosomal (~180 bp) sections. In the TUNEL assay such cells are only weakly labeled. It should be noted that with most TUNEL kits (including APO-BRDU) a positive control cells are provided. They are camptothecin-treated leukemic cells which are expected to have internucleosomal DNA cleavage in about 30-40 % of the cell population (S phase). This control, if found to be TUNEL positive, provides an assurance that the kit is OK. Furthermore, in the case when the control is positive but the investigated apoptotic cells are negative, it provides evidence that DNA fragmentation in the studied cells did not progress into internucleosomal DNA sections. 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. Apoptotic cells are strongly labeled in TUNEL assay only when they have a large number of DNA strand breaks. This is the case when DNA fragmentation is very extensive, which occurs when internucleosomal DNA sections are cleaved. However, in some cell types (often of epithelial or fibroblast lineage) or instances DNA fragmentation stops at the initial step i.e. generating 300 - 50 kb DNA sections, and does not progress into intenucleosomal (~180 bp) sections. In the TUNEL assay such cells are only weakly labeled. It should be noted that with most TUNEL kits (including APO-BRDU) a positive control cells are provided. They are camptothecin-treated leukemic cells which are expected to have internucleosomal DNA cleavage in about 30-40 % of the cell population (S phase). This control, if found to be TUNEL positive, provides an assurance that the kit is OK. Furthermore, in the case when the control is positive but the investigated apoptotic cells are negative, it provides evidence that DNA fragmentation in the studied cells did not progress into internucleosomal DNA sections.
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