The principle of the assay is to evaluate cytotoxicity of the various drugs in HEK293 in the presence and absence of MMHH as an exogenous metabolic system, and in the presence and absence of cofactors.
For the assay, HEK293 cells were trypsinized from the stock cultures and suspended in UCPM. Cell density was quantified using a hemocytometer and adjusted with UCPM to approximately 500000 cells per mL. A volume of 10 uL (containing approximately 5000 cellls) was delivered into each of the wells in the 384-well white plates (treatment plates) employed for the assay.
After a 4-hour culturing duration to allow cell attachment, 10 uL of MMHH supplemented with the designated cofactors were added into each of the treatment wells, followed by 10 uL of the drugs to be evaluated (at 3X of the desired final concentration). The treatment plates were returned to the cell culture incubator for a treatment duration of 24 hours followed by viability determination via quantification of cellular ATP contents.
