Apoptosis inducer - (Nov/07/2005 )
Can anyone suggest a cheap and easily available inducer of apoptosis in A549 cell lines. I would like to use it as control for apoptosis in my cell lines. I am studying some molecules that are killing my mammalian cells (A549) and am interested in studying the mechanism of action.
I use staurosporine as apoptotioc inducer. Its cheap and widely used as apoptotic inducer
I used etoposide (sometimes called VP-16), it's even cheaper than staurosporine.
Ive ordered for etoposide. While I am waiting to get it, could you please let me know your protocol for treating the cells with etoposide and how you score for apoptosis. What assay are u using for studying the apoptosis.
i use etoposide at 1 to 10 µM final in 1 ml of complete medium on 500,000 cells (lymphoma cells)
i prepare an intermediary stock solution at 1 mM in DMSO stored at 4 °C
i obtain 98% of apoptosis at 10 µM after 16 h of incubation (detected by DiOC6(3) and annexin V)
Sorry, took a while...
I used it to induce apoptosis in 293 T cells for a positive control in a screening for PARP in a Western Blot. I used it similar to "dose response" ranging from 5 µM -20 µM. I didn't check apoptosis rate, but got nice PARP cleavage and DNA fragmentation after 16 - 20 hrs.
actually I have the same question, but I am looking for some more specific inducer, since I believe the inhibiting kinases or topoisomerase II influences too many cellular processes other than apoptosis.
So does anyone have suggestions how to induce apoptosis more specifically. Also both substance induce the intrinsic pathway (as far as I know). Any suggestions how to induce the extrinsic pathway? The only thing I heard about is using Fas ligand or antibodies. Maybe somebody has other ideas, anything would help.
Thanks a lot.