Developing a multiplex assay to monitor apoptosis and necrosis using the Cellaca™ PLX Image Cytometer

Abstract

Abstract Apoptosis is a critical pathway for programmed cell death that cancer cells can evade to ensure survival. For pharmaceutical drug discovery, it is important to characterize and compare cancer that can initiate the process of apoptosis, allowing for the identification of potential therapeutic candidates. In this work, we developed and demonstrated a multiplex detection method for monitoring apoptosis and necrosis with Annexin V-APC, Caspase 3/7, and Propidium Iodide (PI) using the Cellaca™ PLX Image Cytometer. First, apoptosis was induced in Jurkat and K562 with staurosporine and monitored over the course of 24 h. Results showed that apoptotic factors and cascades were successfully detected along the pathway from early to late-stage apoptosis, and ultimately necrosis. A clear trend was observed during the first 2 h showing differences of up to 25% between the single Annexin V+ and Caspase-3+ populations in treated Jurkat cells, however, a significant increase in double positive apoptotic/necrotic cells for Annexin V+PI+ and Capase-3+PI+ was not observed until 20 h. Upon further analysis amongst apoptotic populations, Annexin V+ only populations were higher than Caspase-3+ only populations by up to 30% between 0-2 hours. The proposed image-based detection method may provide an effective and efficient tool that allows researchers to better characterize and screen potential cancer therapeutic drug candidates in a high-throughput manner to determine their treatment efficacy.