Multiparametric analysis of cell health status using flow cytometry

Abstract

Abstract Cellular responses to drug treatment can be varied and heterogeneous. Often, a multiparametric approach is necessary to identify cellular pathways that are responding or affected. To maximize the number of parameters analysed per sample, we aimed to combine several functional sensors into one assay. In addition, we set out to streamline the workflow focusing on live cell responses. To this end, we designed a flow cytometry-based detection system centered on a single incubation condition for sensors that have minimal spectral overlap. Analysing Jurkat cells treated with camptothecin, a topoisomerase inhibitor that triggers cellular apoptosis, we were able to simultaneously detect the loss of cells in G2/M phase using the stoichiometric DNA dye, Hoechst 33342; an increase in apoptotic cells with a sensor for activated caspase 3/7 (CellEvent Caspase-3/7 Green); the loss of mitochondrial membrane potential with tetramethylrhodamine, methyl ester (TMRM); and an increase in dead cells as well as cells in late apoptotic stage, using a SYTOX viability dye. We also tested another four-sensor combination that focused on detecting cellular stress. Upon prolonged camptothecin induction, we were able to detect an increase in mitochondrial stress with MitoSox, a sensor that detects mitochondrial oxidative stress; an increase in cellular stress with a CellROX reagent specific for reactive oxygen species. There was also a loss in mitochondrial membrane potential as well as a greater loss of cellular viability. In summary, using a model of cellular response to drug treatment, we performed multiparametric cell status analysis with a combination of cellular detection reagents on a flow cytometer.