How to detect apoptosis?? - (Aug/11/2005 )
Does anybody recommend some convincing methods to detect apoptosis?? thanks a lot.
Can you be more specific? There are many ways to detect/guantify apoptosis, it depends on the tools you have in your lab. Hoechst staining, FACS (PI or Annexin V staining), trypan blue, DNA laddering/fragmentation etc. The choise among them depends on your apoptosis inducing protocol you are using, on your cell line, if you want quantification of apoptosis or just detection and all that.
when apoptosis occurs, dna is degraded between histones. Hence on an agarose gel, you are able to see a ladder of 180bp, and multiples of 180pb (that says 360, 540, 720 etc...)
it also depends on the cell type you're working with. For epithelial cells that express cytokeratin 15, I had very nice results using the cytoDEATH M30 antibody from Roche.
A very good compendium about apoptosis detection techniques can be found here: http://www.roche-applied-science.com/PROD_...N/cell_toc.html
(I hope posting a company related link does not violate any rules of this forum, otherwise please remove)
another method consist of measuring the decrease in mitochondrial transmembrane potential (∆ψm) which is associated with a reduction of the cationic dye DiOC6(3) uptake as seen by flow cytometry
DiOC6(3): 3,3'-Dihexyloxacarbocyanine iodide
this reagent is cheap and experiments reproducible
-Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo (PMID: 7722446 )
-Comparison of DiOC(6)(3) uptake and annexin V labeling for quantification of apoptosis in leukemia cells and non-malignant T lymphocytes from children (PMID: 10679746 )