Anti mip1β pe

An assay to determine NK cell activation (NKA) state based on the expression of surface CD107a and intracellular production of IFNγ and MIP1β was performed as previously described. NK cells were isolated from whole blood from healthy donors using negative selection with RosetteSep (STEMCELL Technologies). Following a pulse with SF162 gp120 (60 μg/ml), T lymphoblast CEM‐NKr cells and isolated primary NK cells were mixed at a ratio of 1:5, and purified IgG, anti‐CD107a‐phycoerythrin (PE)‐Cy5 (BD), brefeldin A (10 mg/ml) (Sigma), and GolgiStop (BD) were added. After a 5‐h incubation at 37°C, cells were first stained for surface markers using anti‐CD16‐allophycocyanin (APC)‐Cy7 (BD), anti‐CD56‐PE‐Cy7 (BD), and anti‐CD3‐Alexa Fluor 700 (BD) and then stained intracellularly with anti‐IFNγ‐APC (BD) and anti‐MIP1β‐PE (BD) after treatment with Fix and Perm A and B solutions (Invitrogen). Cells were then fixed in 4% paraformaldehyde and analyzed by flow cytometry. NK cells were defined as CD3‐negative and CD16‐positive and/or CD56‐positive, and the percent of NK cells positive for each marker was determined.

Natural killer cell (NK) activation and degranulation via CD107a, IFN-γ and MIP-1β detection was assessed via an ELISA-based antibody-dependent natural killer (NK) cell activation assay [52 (link)]. ELISA plates (Thermo Fisher NUNC MaxiSorp flat bottom) were coated with gp140 ConS (200 ng per well) at 37°C for 2 h followed by blocking with 5% BSA in PBS overnight at 4°C. NK cells were isolated from buffy coats from healthy donors with RosetteSep (Stem Cell Technologies) and rested over night with 1 ng/ml IL-15. The next day, after washing the blocked ELISA plates, 50 μl of samples at a 1:25 dilution in PBS were added to each well. Plates were incubated at 37°C for 2 h to allow immune complex formation. Then, 5×10⁴ NK cells with anti-CD107a-PE-Cy5 stain (BD), brefeldin A (5 mg/ml) (Sigma), and GolgiStop (BD) were added to each well and incubated for 5 h at 37°C. NK cells were fixed and permeabilized using Perm A and B solutions (ThermoFisher). Cells were subsequently stained for surface markers with anti-CD16 APC-Cy7 (BD), anti-CD56 PE-Cy7 (BD) and anti-CD3 AlexaFluor 700 (BD). Intracellular staining included anti-IFNγ FITC (BD) and anti-MIP-1β PE (BD). Acquisition occurred by flow cytometry (IntelliCyt, iQue Screener plus). NK cells were defined as CD3- and CD56+. The antibody-dependent NK cell degranulation assay was performed in duplicate across two blood donors.

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.