Enspire multimode plate reader

The detection of FcγRIIIa-mediated signaling was performed using a Jurkat NFAT-luc FCγRIIIa cell line (BPS Bioscience, CA, USA), as described previously [59 (link)]. The target cells were 293T cells expressing Env, which were transfected with Env-expressing plasmid 48 h before the ADCC assay. The target cells were washed with PBS, treated with 0.05% trypsin, and resuspended in RPMI-1640 with 4% FBS at a concentration of 3 × 10⁶ cells/ml. Then, 25 µl of the target cells were incubated with antibodies for 15 min, after which 25 µl of effector Jurkat cells were added at a ratio of 1:1 and were co-cultured for 6 h. The cells were lysed and the firefly luciferase activity was determined with a luciferase assay kit (Promega) and EnSpire® Multimode Plate Reader. The co-culture in the absence of antibody provided background (antibody-independent) luciferase activity. The RLU obtained in the presence of antibody were divided by the background level to calculate the fold change.