Tran, I'm not sure this helps, but many previous studies using TNFalpha to induce apoptotis used actinomycin D, cyclohexaminde or INFgamma to pre-treat the cells. As TNFalpha induces both pro-death and pro-survival signaling, the pre-treatment with these agents acts to block the synthesis of pro-survival factors (NFkappa B, IAP1,2, ect) among other things, thus allowing for the pro-apoptotic factors to complete their task. I always preferred INFgamma as it, along with TNFalpha, are normally found in the circulation during inflammatory pathologies. As camptothecin is a TOP1 blocker, I'm not sure you will get the same effect.

Thank you for your help. There are some studies co-treat TNF alpha and camptothecin to induce apoptosis, but in another cell lines, not HEK293.

I will try TNF gamma as your suggestion.

I used PARP as a marker of apoptosis, and checking its increasing after treatment.

And I also treat only TNF alpha and checking ikB alpha for survival pathway.

Could you give me some experience about it?

Best.

Gin

Previously I treated MEFs with 20 ng/ml interferon-gamma for 18-24 hrs and then treated with 40 ng/ml TNFalpha. 30 min after TNFalpha, IkBalpha was mostly degraded. By 24 hrs the cells were 50% dead as determined by TUNEL. One might assume PARP to be cleaved at this point as well, but I did not measure this.

Tran Hue Vy An William Blalock

I tried treating human fibroblasts with IFN-y and TNF-a. What are the concentrations that worked for you Tran? I tried with 40 ng/ml IFN-y and 20-80 TNF-a, but they only died a little bit.

Hwy Tran, did the protocol with INFgamma worked? I'm trying to induce apoptisis on my HEK293 cells, but so far I've seen only 20% cell death. Could you share your protocol with me?