Caspases are involved in the initiation and execution of apoptosis. They are

apoptosis initiates (caspase-2, -8, -9, -and -10), and apoptosis executioners (caspase-3, -6, and -7). There are several drugs that's induces these caspases, especially anti-cancer drugs. In simple you can try drugs like ionomycin, staurosporine, Paclitaxel, bleomycin are commonly used drugs in laboratories for inducing apoptosis. Even exposure to defined doses of UV radiations induce apoptosis in cells.

Regarding inhibitors of apoptosis, Caspases are regulated by members of the inhibitor of apoptosis (IAP) family such as cIAP-1, cIAP-2, XIAP and also Survivin and Livin. IAP activity is modulated by DIABLO/SMAC. Use of drugs that induce IAPs or use of compounds that inhibits mediators of apoptosis including c-Myc, Bax, p53, tBid, and BCL may be helpful.

Vinod Nadella thank you for the answer, but I meant something else.

I conduct an experiment with an apoptosis-inducing antibody. That antibody is Rituximab. The question is, which factors can inhibit rituximab-induced apoptosis? Or stimulate the process? During every step of experiment something could happen that, for example, inhibits biding of given antibody to the receptor. Or removes the receptor from the cell surface (my only idea I've mentioned before - trypsin). Or inhibits forming of lipid rafts. Or inhibits caspases, but we are not aware of the fact (I mean - we're not using designated caspase inhibitors. We've got some agent that inhibits caspases, unknown agent, in buffer, or in medium).

I'm looking for agents that could be the reason for these.

Rituximab induce apoptosis in cells expressing cell surface CD20 in a MAP-kinase phosphorylation dependent manner and depends on p38 activity. So, technically, and to be more specific, use of p38 inhibitors will significantly reduce anti-CD20–induced apoptosis.

Worth reading below printed articles for better understanding..

https://ashpublications.org/blood/article/99/4/1314/110007/The-chimeric-anti-CD20-antibody-rituximab-induces

Article Inhibition of human B-cell lymphoma by an anti-CD20 antibody...

https://www.nature.com/articles/1206939

Vinod Nadella I see. I've read these papers and I agree. I just mean that I don't want to inhibit rituximab-induced apoptosis on purpose.

I'm more troubleshooting here. I do not want to introduce other agents to the experimental system. I want to analyze the process and come up with ideas, factors, that could be present for example in medium, buffer and could influence apoptosis process. Like prolonged incubation with trypsin in adherent cells, which could digest CD20, being on the surface of cells.

It is a difficult question. I guess you want to know if some kind of error accidentally rescues the mab-induced apoptosis or whatever, am I right? When CO2 sensor goes down (when the sensor being wet), the medium pH will change. I'm sure low pH quickly kill the cells, however, if the antibody became fragile, the result would be different. Lower temperature would happen when the incubator goes wrong. Lower temp inhibit the cell proliferation, however, the membrane traffic will also change at very low temp, which will change the internalization of cell surface receptor. If there is a road construction next to the lab, the vibration might shake the culture dish badly. The cells would probably be fine, but the antibody reagents might be damaged.

Hi Natalia

Your model system is using rituximab to induce apoptosis and your question is which all factors can inhibit rituximab-induced apoptosis?

Going back to the mechanism of rituximab-induced apoptosis, as I already mentioned, it is via. cell surface expressed CD20 in a MAP-kinase phosphorylation dependent manner. Inhibiting the expression of Cd20 or blocking the CD20-rituximab interaction or inhibiting MAP-kinase signaling pathway will meet your needs. However, you say that you do not want to introduce other agents to the experimental system.

In this case, u think prolonged trypsinization may meet your purpose thinking that it may lead to chop off cell surface Cd20. But, you also got to think that prolonged trypsinization will kill cells not just by mean loss of CD20 but by varied other mechanisms. Use of trypsin may sound relevant with adherent cells, but what about non-adherent and semi-adherent cell types. So, ur idea may not be global !!

If you think of altered cultural conditions like changes in culture media, temperature etc., that may or may not be mediated via Cd20-rituximab, which you got to check.

By the way, what is ur cell model?

If you think I still haven't got your question properly, I am extremely sorry and I am also looking forward for a better inputs

Vinod Nadella thank you for your time and all the answers! So let me get this straight: it's a literature project. I am going to perform rituximab-induced apoptosis detection experiments, but for now, I have to analyze the process in theory. And I have to come up with ideas, with factors, that could lead to better performance and the ones that could negatively influence my potential results. I know the theory of the process, but now - more practical questions arise. And in case of trypsin - yes, that's true - trypsin is not a global idea. Only in case of adherent cells. But right now, I have to think global and analyze all the possibilities in case of every cell culture type.

I was also thinking about cholesterol concentration. I know that it's important for lipid raft formation, but too high cholesterol concentration can make the membrane rigid, which is also not good. An obvious stimulant is a cross-linker. And I'm looking for some other ideas, because it's all I came up with, due to the fact, that I don't know experimental aspects of apoptosis detection.

I basically ask what could go wrong and what do we usually do to obtain better results (e.g. crosslinker).

your subject is very interesting for me but I now try to clarify the type of cell death not just apoptosis necrosis,, necroptosis and signaling pathway

congratulations to chose this subject Natalia Urbańska