Anti cd16 apc cy7

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.

To compare the surface expression of CD16, CD69, and CD161 on NK cells at the whole range and single-cell level, PBMCs of healthy donors were pre-incubated with cytokines for 12 h and then washed with PBS. All samples were stained with anti-CD3 eFluor 450 (ebioscience, clone UCHT1), anti-CD16 APC-Cy7 (BD, clone 3G8), anti-CD56 PE-Cy7 (BD, clone B159), anti-CD69 APC (BD, clone FN50), and anti-CD161 PE-Cy5 (BD, clone DX12). All antibodies were purchased from BD Biosciences (San Diego, CA, USA) except anti-CD3 from eBiosciences (San Diego, CA, USA). NK cells were defined as phenotype of CD3⁻CD56⁺ and the frequencies of CD16, CD69 and CD161⁺ NK cells were analyzed. CD16/CD69/CD161 expressions on NK cells were acquired on BD FACS Fortessa (BD Biosciences, USA) and then analyzed by FlowJo software (Treestar, Ashland, OR, USA).
The prepared samples were also run on ImageStream χ MarkII system (Amnis Corporation, Seattle, WA, USA) to acquire the data of CD16/CD69/CD161 expressions on single-cell. Twenty thousand events were collected for each sample and single-color control was used to create a compensation matrix to correct for spectral overlap. Collected data were analyzed using IDEAS 3.0 software (Amnis Corporation, Seattle, WA, USA).

ELISA plates were coated with antigen at 300 ng/well and incubated for 2 h at 37 ˚C. Plates were blocked with 5% BSA in PBS overnight at 4 ˚C. The next day, 100 μL of diluted sample were added to the plates. Plates were incubated for 2 h at 37 ˚C to form immune complexes. During the incubation, human NK cells were isolated from buffy coats using RosetteSep NK cell enrichment kit (StemCell Technologies #15065) and Ficoll separation. After the incubation, NK cells were added to the plates at 5 × 10⁴ cells/well in R10 supplemented with anti-CD107a PE-Cy5 (1:80 dilution), GolgiStop and Brefeldin A (BD Biosciences,#554724, #555802, Sigma #B7651). Plates were incubated for 5 h at 37˚C. Following the incubation, NK cells were stained for surface markers with anti-CD56 PE-Cy7, anti-CD16 APC-Cy7 and anti-CD3 Pacific Blue (1:200 dilution) (BD Biosciences, #557747, #557758, #558124). NK cells were fixed and permeabilized with Fix&Perm cell permeabilization kit (Invitrogen). Cells were incubated with anti-MIP1β PE (1:200 dilution) and anti-IFNγ FITC (1:80 dilution) (BD Biosciences, #550078, #340449) to stain for intracellular markers. Cells were acquired on a Stratedigm 1300EXi cytometer.