Detection of Cellular Apoptosis Using Calcium Free Fluorescent Tagging and Imaging Flow Cytometry 2165

Abstract Description Apoptosis is a normal physiological process for cell elimination during tissue homeostasis. One of the hallmark characteristics of apoptosis is the membrane-bound translocation of phosphatidylserine (PS) to the external cellular environment. To ensure that assays are accurate and robust, it is important to verify cell viability, as well as identify and exclude apoptotic and dead cells. Conventional flow cytometry commonly detects cell surface PS in apoptotic and dead cells with annexin V (AV) staining in the presence of calcium (Ca2+) rich buffer. However, the Ca2+ rich buffer may cause cell clumping in the sample, thus, it can be quite difficult to get reliable flow cytometry analysis results. In addition, since the AV-stained cells cannot be fixed after staining, any downstream process such as intracellular staining requiring fixation cannot be performed. A class of fixable, fluorescent cellular tags (Tag) have been developed recently for detecting PS in apoptotic and dead cells using Ca2+ free buffer. This study evaluates the feasibility of using a fluorescent tag for PS detection in several apoptosis-inducing conditions such as Camptothecin (CPT) treatment and starved cell cultures. Samples were stained and data collected on a Cytek® Amnis® ImageStream®X Mk II imaging flow cytometer. Our results demonstrate that these tags are suitable to detect apoptotic and dead cells in a Ca2+ free environment with imaging flow cytometry. Topic Categories Technological Innovations in Immunology (TECH)