To date, multiplexing cell-based assay is essential for high-throughput screening of molecular targets. Measuring multiple parameters of a single sample increases consistency and decrease time and cost of assay. Functional assay of living cell is useful as a first step of multiplexing assay, because live-cell assay allows following second assay using cell lysate or stained cell. However, live-cell assay of drug resistant cells that are highly activated of drug efflux mechanisms is sometimes unstable or difficult; for example, the method of measuring colored formazan products by living cells does not show correlation between the amount of formazan and the cells numbers. To this end, more reliable method to allow live-cell assay is anticipated.
We described here the protocol of live-cell luciferase assay as a first step of multiplexing cell-based assay of drug-resistant cells which expresses firefly luciferase. This method has several advantages; 1) In growth assay of P-glycoprotein-overexpressing multidrug-resistant cells, firefly luciferase bioluminescence from live cells correlates with the cells numbers; 2) Live-cell assay allows performing second assays, such as a caspase assay, Hoechst staining, and a cell-direct real-time RT-PCR, using the same cells. If necessary, it is possible to continue the cell culture replacing the assay solution with fresh medium; 3) Live-cell luciferase assay is available for other mammalian cells besides drug-resistant cells.