NK Cell Degranulation and Cytolysis Assay

A modified ELISA-based assay for the detection of CD107a as a surrogate marker of NK-cell-mediated degranulation and cytolysis was performed as previously described (40 (link)). Briefly, a 96-well ELISA plate was coated overnight at 4°C with recombinant protein. Purified IgG was added to each well, and the plate was incubated at 37°C for 2 h. HIV-negative plasma samples or medium alone was used as a negative control, while HIVIG (pooled HIV immunoglobulin G) (NIH AIDS Reagents Program) was used as a positive control. A total of 5 × 10⁴ NK cells enriched via negative selection from healthy blood donors (RosetteSep; Stemcell Technologies) were added to each well in the presence of brefeldin A (BioLegend), Golgi stop, and anti-CD107a-PE-Cy5 (BD Biosciences). Plates were incubated for 5 h at 37°C with 5% CO₂. Cells were then stained with anti-CD3-AlexaFluor700, anti-CD56-PE-Cy7, and anti-CD16-allophycocyanin (APC)-Cy7 (BD); fixed with Perm A; permeabilized using Perm B (Invitrogen); and stained with anti-IFN-γ–APC and anti-MIP-1β–PE (BD). The cells were fixed with a 2% paraformaldehyde solution and analyzed by flow cytometry.