The need for characterizing apoptotic processes occurs throughout the drug discovery process – from primary screening to toxicity profiling. Apoptosis is a tightly regulated cell death program that can be executed by cells that are no longer physiologically necessary. It is often triggered as a response to extrinsic factors or inhibited for survival as in the case of many cancer cells. Cell death cascades are complex and dynamic, underscoring the importance of a multi-parametric approach to assess apoptosis. This underscores the need to conduct robust and reliable cellular assays at higher densities and with smaller sample sizes. As such, technological advancements such as High Throughput Flow and low-volume liquid handling have become critical components of methods assessing apoptosis. Using the Intellicyt iQue HD Screener and MultiCyt® 4-Plex Apoptosis Screening Kit in conjunction with the Labcyte Echo liquid handler, we were able to simultaneously detect 4 different apoptosis endpoints in Jurkat cells in both 384- and 1536-well formats.

In this study, Jurkat cells were treated for 24 hours with known apoptosis inducing agents: staurosporine, nocodazole and camptothecin. After treatment, cells were labelled for one hour with a no-wash / single step addition of fluorescent markers for caspase 3/7 activation, Annexin V binding, cell viability, and mitochondrial membrane depolarization. Sub-microliter volumes of compounds and dye were transferred to the 384- and 1536-well cell plates with the Echo liquid handler. The results show equivalent potency estimates for the compounds tested in both plate formats and correlate to previously reported activity for the biomarkers measured.