The IC50 of my drug in my cell line (MSTO) is 25 nM.
I recently did a Caspase-3 assay using 2.5 nM, and the relative caspase-3 activity increased to 300 compared to the untreated control. This is a similar increase to that I've seen in another cell line (Ren) where the IC50 is 2.5 nM!
What could be the reasons behind this? Can cells be viable according to an MTT while going through apoptosis?
Note: My drug is an ADC that binds to mesothelin. Ren cells expressed mesothelin, MSTO don't (well, 10 % ish).
Hello Ms. Aimee
See MTT cell viable assay is an assays which specifically shows mitochondria based viability i.e. when cells uptake MTT salt it undergoes reduction to MTT formazan under succinate hydrogenase action. Other than that if you take an LDH assay it gives you whether the cell has gone through necrosis, as cells after membrane instability the LDH is released which reduces lactate to pyruvat. So you see viability study can show various results, Therefore your results are still incomplete, you should check the cell viability of the cells you used using other viability studies such as XTT, NRU, LDH and methylene blue staining and define cell death path and along with it try to test the protein signalling pathways then only I can give a proper answer to your question and even you could give it yourself also..
Best wishes
Raunak Saha
Caspase 3 activity is just one of the proteins contributing to apoptosis.
How you measured Caspase 3 activity, using elisa, in vitro or in cells?
If in vitro, it is very normal because of a lot of factors like delivery, metabolism, distribution.
Caspase 3 activity is measured using a plate reader based assay from Promega.
So from what I understand from the replies, the cells themselves may be undergoing apoptosis, resulting in high caspase 3 activity, but still have functional mitochondria, leading to higher than perhaps is true cell viability?
you can elongate the incubation time like 24h or 48h more.
or do colony formation assay using treated cells, from which you can distinct if your current cells is good . inform me when you need protocol.
Ms. Amie
I am in total satisfaction with Mr.Xinlai Cheng. And apoptosis can triggered by multiple protein signalling pathways such as MAPK (JNK,ERK, and P38), tumer suppressant protein, Bax, BCL2, procaspase-9, etc. So you should isolate the proteins and do western blotting of this adforementioned proteins to confirm apoptosis otherwise your results are in doubt as they are partially done as cells might have gone apoptosis or necrosis.
The cells themselves may be undergoing apoptosis, resulting in high caspase 3 activity, but still have functional mitochondria, leading to higher than perhaps is true cell viability? Yes in our case we saw that multiple assays shows multiple cell viability so you cannot confirm that apoptosis is for sure. Hence, to be sure you have to do protein signalling tests, otherwise there would be a doubt regarding the results for sure.
Best wishes
Raunak Saha
Dear Amiee,
Altough I am not sure about your data. I can only give some idea after you explain all the data in detail regarding cell death.
However apoptosis is not the only cell death mechanism. You may read about Pyroptosis, Necroptosis, ferroptosis and their relation with cell death, mitochondrial activty, caspase3 activity etc and designe your experiment acodingly to understand more about your findings.
All the best,
Diptiman
Be aware of pitfalls of the MTT assay resulting in a wrong estimation of viability. Stepanenko AA, Dmitrenko VV. Pitfalls of the MTT assay: Direct and off-target effects of inhibitors can result in over/underestimation of cell viability. Gene. 2015 Dec 15;574(2):193-203. doi: 10.1016/j.gene.2015.08.009.
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