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Background Natural Killer (NK) cells are critical components of the immune system that have multifaceted roles in protecting against disease. NK cells recognize and facilitate the destruction of various pathogens and are proposed to have further implications in the control of cancerous cells. CAR-T therapy is a recent FDA approved treatment which utilizes T-cells engineered to express specific CARs (Chimeric Antigen Receptors) to target cancerous cells. CAR-T therapy is largely limited to blood-based cancers, most commonly leukemia, and has little efficacy on solid tumors. Although this is the FDA standard, CAR-T includes other limitations and risks including, a secondary cancer risk derived from the longer lifespan of T cells, limited dosages of the treatment, and the need for autologous T cells to avoid complications associated with MHC restriction.Methods Our project explores the use of iPSC derived NK cells as effectors for CAR-NK therapy with the hope of overcoming many of these limitations. The shorter lifespan of NK cells may help overcome CAR-T limitation as they can be programmed to kill cancerous cells and naturally die off, eliminating the risk of a secondary cancer. In addition, CAR-NK can be applied in an allogenic manner, expanding therapy access. The purpose of this research was to optimize surface staining of cell lines, apoptosis marker staining via Annexin V and apoptosis induction parameters in a flow cytometry-based killing assay. First, K562 cell line was characterized for several surface antigen expressions via fluorescently labeled antibodies (CD45 and CD71). 1 Once K562 characteristic surface staining was established, apoptosis induction conditions were optimized using ultraviolet light exposure, and apoptotic events were detected using Annexin V staining.2 3 Results We further explored various UV exposure times to test optimal apoptosis induction after confirming the efficacy of antibodies CD45 and CD71. This allowed us to then confirm optimal UV exposure (45 minutes) and post-exposure times (30 minutes) for K562 cell lines. Annexin V was utilized to confirm apoptotic events across various optimal induction times for these cell lines.Conclusions These parameters provide us with a repeatable set of conditions for apoptosis induction which will serve as a positive control in the future development of an NK-based killing assay using K562 targets. 4 Future directions will implement this killing assay in engineered CAR-NK effector cells.Acknowledgements I would like to thank Dr. Goicochea for his time and mentorship over the past year, in addition to the Stevenson community for their support and resources in this project.References Karagiannis TC, Wall M, Ververis K, Pitsillou E, Tortorella SM, Wood PA, Haloom Rafehi, Khurana I, Maxwell SS, Hung A, Jitraporn Vongsvivut, Assam El-Osta. Characterization of K562 Cells: uncovering novel chromosomes, assessing transferrin receptor expression, and probing pharmacological therapies. Cellular and Molecular Life Sciences 2023;80(9). https://doi.org/10.1007/s00018-023-04905-6Annexin V Staining - US. www.thermofisher.com. https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/apoptosis/annexin-v-staining.html. BestProtocols: Annexin V Staining Protocol for Flow Cytometry | Thermo Fisher Scientific - US. Thermofisher.com. https://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/protocols/annexin-v-staining-flow-cytometry.html (accessed 2024-12-06)Crowley LC, Marfell BJ, Scott AP, Waterhouse NJ. Quantitation of apoptosis and necrosis by annexin v binding, propidium iodide uptake, and flow cytometry. Cold Spring Harbor Protocols 2016;2016(11):pdb.prot087288. https://doi.org/10.1101/pdb.prot087288Saetersmoen ML, Hammer Q, Valamehr B, Kaufman DS, Malmberg K-J. Off-the-shelf cell therapy with induced pluripotent stem cell-derived natural killer cells. Seminars in Immunopathology 2019;41(1):59–68. https://doi.org/10.1007/s00281-018-0721-x