Use of External Metabolising Systems in the Neutral Red Assay

BALB/C 3T3 mouse fibroblasts or CHO cells grown in 96-well microtitre plates were used for the neutral red viability assay. The cytotoxicity and ability of microsomes or an S9 mixed function oxygenase to activate cyclophosphamide were compared. A pronounced cytotoxic effect of both activation systems was found using CHO cells, but not using 3T3 cells. 3T3 cells were more sensitive than CHO cells to the active metabolites of cyclophosphamide. Cyclophosphamide was more efficiently converted to toxic metabolites by microsomes than by the S9 mix. A concentration of microsomes corresponding to 0.032mg protein per ml of medium was shown to be optimal for the activation of cyclophosphamide in both cell lines.