Abstract 356: Developing a multiplex assay to monitor apoptosis and necrosis using the Cellaca® PLX Image Cytometer

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 therapeutics (i. e. , small molecules, antibody drugs, cell therapies) 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, Caspase 3/7, and Propidium Iodide (PI) using the Cellaca® PLX Image Cytometer. First, apoptosis was induced in Jurkat and K562 cell lines with staurosporine over the course of 24 h, and were monitored at 0, 1, 1. 5, 2, 4, 20, and 24 h timepoints. Samples were stained with Hoechst 33342 (total dye), Annexin V-APC (early-stage apoptosis), Caspase-3 488 (late-stage apoptosis), and PI (necrosis) at each timepoint and evaluated using the Cellaca® PLX. 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. Conversely, K562 cells did not exhibit a notable change in apoptotic and necrotic populations due to low sensitivity to staurosporine. The proposed image-based detection method using the Cellaca® PLX may provide an effective and efficient tool for rapid and reliable simultaneous detection of early, late-stage apoptosis, and necrosis. This may allow researchers to better characterize and screen potential cancer therapeutic drug candidates in a high-throughput manner to determine their treatment efficacy. Citation Format: Joydeep Mukherjee, Mackenzie Pierce, Yongyang Huang, Allen Lin, Leo Li-Ying Chan. Developing a multiplex assay to monitor apoptosis and necrosis using the Cellaca® PLX Image Cytometer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 356.