Anti cd16 apc cy5

ELISA plates were coated with 2 ug/mL of spike, incubated for 2 hours at 37°C, washed three times with PBS and blocked overnight at 4°C in 5% BSA in PBS. Human NK cells were isolated from peripheral blood (MGH Blood Bank) using RosetteSep kit (Stem Cell Technologies) followed by Ficoll separation to isolate cells. NK cells were maintained overnight at 37°C in RPMI media with 10% fetal bovine serum, L-glutamine, HEPES, penicillin/streptomycin and IL-15. Blocked plates were washed three times with PBS, and diluted serum (1:50) and diluted breastmilk (1:5) were added to the coated ELISA plates. Plates were incubated for 2 hours at 37°C. After the incubation, plates were washed three times with PBS, and NK cells were added at a concentration of 2.5 × 10ˆ5 cells/mL in media supplemented with GolgiStop (BD), Brefeldin A (BFA, Sigma Aldrich) and anti-CD107a PE-Cy5 (BD) and were incubated for 5 hours at 37°C. Following the incubation, NK cells were stained for surface markers with anti-CD3 PacBlue (BD), anti-CD16 APC-Cy5 (BD), and anti-CD56 PE-Cy7 (BD). After staining, cells were fixed using the FIX&PERM A/B kit (Life Tech) and stained for MIP-1b (anti-MIP-1b PE, BD). Fluorescence was acquired using an iQue (Intellicyt). NK cells were gated as CD56+/CD16+/CD3- and NK cells activity was determined as the percentage of NK cells that were positive for CD107a and MIP-1b.