A high-throughput image cytometry-based screening method for the detection of IL2-induced peripheral blood mononuclear cell-mediated cytotoxicity

Abstract

Abstract Cell-mediated cytotoxicity assays have been an important functional test for investigating the cytotoxic effect of immune effector on target cancer cells. Cytotoxicity assays have traditionally been performed using the 51Chromium (51Cr) release assay, which involves labeling the tumor cells (target) with radioisotopes. The 51Chromium release assay is highly hazardous, time-consuming, and can only acquire end point readout. Furthermore, it can generate inconsistent results due to batch variation, ability of the target cell for 51Cr uptake, and low sensitivity. In this work, we demonstrate a novel high-throughput cytotoxicity screening assay using the Celigo imaging-cytometry method. First the live K562 target cells were stained with Calcein AM, and were co-cultured with PBMCs from two healthy donors in a flat- bottom 96-well plate in the presence or absence of IL-2. Direct cell counting of Calcein positive cells was implied to calculate the changes in number of live target cells in the same well from T = 0 to T = 4 hours. Thus, accurate cell-mediated killing was determined using the “self-referencing” calculation, which could be a more direct method for assessing cytotoxicity than 51Cr release. The donor PBMCs were added to each well with different Calcein-stained target cells at Effector-to-Target (E:T) ratios. The 96-well microplate was scanned and analyzed using the Celigo image cytometer at different time points to measure the % lysis of target cell. The proposed image cytometry method can scan and analyze in 7 min, which can be utilized to perform high-throughput screening of potential antibody- or chemical-based cancer drug, which could be a more efficient method for academic, industry, and clinical research.