Guys. What should I expect when I run LDH assay for nocodazole treated fibroblasts? An increased cytotoxicity or profile similar to cells+medium.. Bcoz I know that nocodazole induces mitotic arrest. However, the cell viability will not be altered for some time when the cells are arrested..
Btw, I treated cells with 1:100 dilutions of nocodazole for 24h and ran the LDH assay
Be careful with the LDH assay. Some kits do not provide reliable results when you follow the protocol (sometimes the protocol is not well written). Do not use end point but use different times and get the amount of LDH by calculating the enzymatic activity (difference in "color"/time). Also consider that if you add double the amount of extract you should get double activity. In summary optimize the assay before using it to get conclusions.
We studied nocodazole (and many other drugs) cytotoxicity in transformed human hepatocytes and found inhibitiion of cell proliferation at 0.1 uM, with nuclear shrinkage (indicator of apoptosis) at 0.4 uM. Mitochondrial activity and calcium homeostasis were adversely affected only at 25 uM. We published this work in Archives of Toxicology in 2006, vol 80 p 580, a copy of which can be gotten through RG.
@Dr. Brien : Thank you for your insights sir. I will definitely have a look at your article and reach you when needed.
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