Ebola ADCC Assay for VLP Cytotoxicity

An Ebola ADCC assay previously developed in our laboratory (60 (link)) was used to assess the cytotoxic potential of Ebola VLP-exposed cells. Briefly, 293 Trex cells (target cells), stably transfected with luciferase and EBOV GP genes, were induced with 2.0 μg/mL of doxycycline for 24 hours. Cells were harvested, washed, and mixed with PBMCs (effector cells), either unexposed (control) or those exposed to Ebola VLPs for 48 hours, at different effector/target cell ratios (10:1, 5:1, and 2:1) in complete media in 96-well plates. All the wells received 1:200 diluted rhesus macaque anti-EBOV GP plasma (71 (link)). Plates were incubated at 37°C, 6% CO₂ for 6 hours. Following incubation, cells were washed thrice with PBS and cell pellets resuspended in 100 μL/well of complete media. Cells were lysed with the addition of 100 μL/well of Britelite Plus luciferase substrate (PerkinElmer), and luciferase activity (relative luciferase units, RLU) was measured on a BioTek Synergy HTX reader. Percentage target cell killing was calculated by the following formula: ([RLU_control – RLU_{anti-EBOV GP plasma}] × 100)/RLU_control.