Real-time Caspase 3/7 measurement of suspension and adherent cells using the Celigo image cytometer

Surekha Bonasu, Sarah Kessel, Leo L Chan, Jean Qiu, Dmitry Kuksin

A novel assay has been developed for real-time staining and detection of active caspases 3 and 7 in apoptotic cells using the Celigo, a micro-well imaging system. Traditionally, this type of apoptosis assay is an end point assay and is performed using fluorescently labeled Annexin-V. However, by coupling this image cytometry system (Celigo) developed by Nexcelom Bioscience LLC (Lawrence, MA) with live-cell dye for caspase 3/7 we are able to image and analyze adherent and suspension cell lines without cell perturbation. Since apoptotic cells are often fragile and can be easily washed off the plate, performing an in-the-plate wash-free assay for the detection of caspase activity is of significant value to the research community. Because the image cytometer is capable of capturing bright-field and fluorescent images as well as perform gating operations based on fluorescence intensity, it is possible to not only carry out live continuous monitoring by detecting a green caspase 3/7 signal but also perform an end point assay by counterstaining the cells with Hoechst and thereby determine a percent nucleated cells that are apoptotic. Here we successfully demonstrate performing real-time continuous monitoring and end-point caspase 3/7 assays using adherent MDA-MB-231 and suspension Jurkat cells stained with Nexcelom ViaStain TM Caspase 3/7reagent in a 96-well format and imaged on the Nexcelom Celigo image cytometer. This combination of live
cell staining and real-time image acquisition provides researchers a unique tool for examining dynamic apoptotic events in a micro-well environment