Hello everyone!
MTT was performed after applying DEHP to HeLa cells. According to MTT results (24 hours of exposure), cell viability is significantly reduced at concentrations of 100 micromolar and above. In the 1-30 micromolar range, viability is almost the same. Next, I will check SOD, CAT, GPX levels with ELISA. What concentrations should I use in ELISA? Should I include IC50, IC30 or IC20?
I would be glad if you help me.
Thanks in advance!
Hi Naile Merve Güven
You could include five concentrations for SOD, CAT, GPX levels with ELISA.
Include IC50 and two concentrations below IC50 and two concentrations above IC50.
Best.
GPX requires a source of GSH to function, plus you don't want the external environment to become depleted in thiols and rich in disulphides. Therefore I would include G6P, G6PDH, GSH and glutathione reductase for the GPX to function for any extended period of time. SOD I personally question it's importance and the 'toxicity' of superoxide itself (see link)
Article Erythrocuprein, also Known as Superoxide Dismutase, Is a Hyd...
Catalase functions well just added to the media, but works better with low concentrations of H2O2 when it can function as a peroxidase. Other oxidants of concern include Fe(IV) species.
Article Hemoglobin Is Not a Biological Fenton Reagent
In my experience many cells exhibit a threshold response, in terms of the observed toxicity, when ROS production (could use systems such as Xanthine/Xanthine oxidase, or glucose/glucose oxidase) exceeds ROS removal rates by the cells. This phenomena also observed by others.
Thesis Nitrofurazone and Hydrogen Peroxide Metabolism in Trypanosoma brucei.
Good luck.
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