Apoptosis detection - Apoptisis detection (Apr/11/2002 )
I have a real problem with the detection of apoptosis in cultured cells.
I detect positive results by many experiments (TUNEL, electronic microscopy.) vs a negative control. I decided to detect DNA fragmentation on agarose gel (BET staining), but surprisingly it was really positive for the negative control cells and not for those of interest (no possible inversion of cells!).
maybe you compare various methods eg FACS-sub G1 DNA peak in PI-staining, staining with annexinV or apo 2.7 assay (detects mitochondrial membranprotein) with FACS??
I don't know if this helps but the DNA fragmentation gels don't work for all cell types. For instance I also use HL-60 cells treated with staurosporine as a positive control as this always shows nice laddering and you know if your treament has work or if it is the wasy you have prepared your samples. Also the tunel assay is not totally specific to apoptosis as some types of necrosis will be tunel positive. The best and most conclusive method to detect apoptosis is to use an assay detecting caspase activity. Hope this helps.