MICROCAT: A Novel Cell Proliferation and Cytotoxicity Assay Based on WST-1

The MICROCAT (96-well microtest plate Consecutive Assay Testing) research method was developed to accurately measure drug effects in vitro with small numbers of cells (10 3 -10 4 cells/culture). Standardization of MICROCAT using rabbit surgically explanted primary eye cells (Tenon’s capsule fibroblasts, RTCF) is described. Results are shown for TCF treated with mitomycin C, rapamycin, and c-myc antisense oligonucleotides. The principles of non-destructive testing were employed; a series of three viable procedures were performed sequentially and repeatedly (i.e., daily) on the same microculture without harming the cells. The first test performed was the WST-1 colorimetric test, which measures cell metabolism (dehydrogenase activities). This was followed by Trypan Blue dye exclusion test for cell viability (cell membrane activity) and then by photographic recording to obtain information on cell number, size, and morphology. An optional final procedure was histological staining with a modified Wright’s Stain prior to photographic recording. MICROCAT improved the accuracy of our experimental results by minimizing the effect of inter-culture variations, which might otherwise mask important drug effects. Other advantages include efficient use of sample material, reduced drug testing costs, relatively rapid results, and increased validity (through the use of normal cells derived from relevant tissue). Methods Cell culture