Hi All,
I'm measuring cell toxicity in different cell lines against aggregated protein solutions. I'm performing various cell viability, toxicity, and ROS level assays. Except for these, what kind of parameters (or metabolites) can I detect to examine toxicity/alterations in cells?
Thank you.
Fromm your experimental setup it seems you are inducing toxicity in various cell lines and trying to analyze cell viability, ROS levels etc. I would suggest you to analyze Nitric oxide (NO) level along with ROS to confirm whether your protein is inducing stress-mediated cell toxicity. Once it is confirmed that ROS and/or NO are elevated, then you can check antioxidant enzymes like SOD, Catalase etc to correlate the cellular antioxidant enzymes with ROS as well as NO level that may signal downstream pathways to initiate cell toxicity including apoptosis, autophagy, necroptosis ans well as necrosis depending upon the extent of oxidative stress insult.
Once you finish the above parameters then you can check the pro-apoptotic and apoptotic markers such as p53, Bax/BCl2, upstream as well as downstream caspases (Caspase 9, Caspase-3 etc) and DNA damage by agarose gel electrophoresis. Its always better to have morphological changes at the level of cell in vitro (I mean morphological apoptotic features such as blabbing, shrinkage, cytoplamsmic fragmentation etc) that can be very useful to correlate the .biochemical well as molecular data as described above.
Although there are several other important parameters that could be analyzed but you have to see that whether your lab facilities/resources allow you to conduct proteomic and genomic level analysis. Good luck
I thought about cell death assays (autophagy and apoptosis), but I didn't consider nitric oxide before. It may be useful for our research. Also, morphological alterations can be observed as you said. Thank you so much for your recommendations!
Alkaline phosphatase assay (ALP) also can be explored for cell death or growth.
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