Anti cd16 pe

High-binding ELISA microplates were precoated with GNL prior to the immobilization of 5 μg/mL GPC-I53-dn5B, incubated at 4°C overnight, washed with 1X Tris-Buffered Saline, and blocked with 1% BSA in PBS. PBMC were obtained from leukapheresis products of healthy donors by Ficoll-Paque density gradient following the manufacturer’s protocol. NK cells were enriched by positive selection from human PBMCs using Miltenyi’s CD56 MicroBeads following manufacturer’s protocol and stimulated with 10 ng/mL IL-15 in Iscove's Modified Dulbecco's Medium supplemented with 10% FCS and 100 U/mL penicillin/streptomycin. Stimulated NK cells were incubated at 37°C overnight. On the day of the experiment, a 1:50 dilution of guinea pig serum in 1% BSA in PBS was added onto the well and incubated for 1 h at 37°C. NK cells were added onto the plate at 35,000 cells per well and incubated for 3 h at 37°C. After incubation, NK cells were transferred into a 96 well V-bottom microplate and stained with anti-CD16-PE (Biolegend) at a 1:1000 dilution (CD16 is a marker for NK cell activation⁶² (link)). As a positive control, phorbol myristate acetate (PMA) (50 ng/mL) and ionomycin (500 ng/mL) was added, and no serum wells was used as a negative control. Samples were read using a BD FACSymphony A1 Cell Analyzer.

Samples of EDTA anticoagulated peripheral blood (6 mL) were collected from control or SARS-CoV-2–infected CMs. Then, diverse cell populations were measured by flow cytometry according to the manufacturer’s instructions with the following monoclonal antibodies: anti-CD3-PE-Cy7 (BD Biosciences, 557917), anti-CD4-APC (Biolegend, 317415), anti-CD8-PE (Biolegend, 301008), anti-CD20-PE (Biolegend, 302306), anti-CD16-PE (Biolegend, 302007), and anti-CD56-APC (Biolegend, 318309). Samples were analyzed on a BD FACS Canto II flow cytometry system (BD Biosciences). For blood routine analysis, anticoagulant venous blood collected at indicated time points were subjected to five classification hematologic analyzer (Mindray, China).

For flow cytometry, we used anti-CD3 FITC (BioLegend, USA, Cat#300440) and PerCP (BD Biosciences, USA, Cat#347344), anti-CD56 FITC (BioLegend, USA, Cat#318304) and APC (BD Biosciences, USA, Cat#555518), anti-CD16 PE (BioLegend, USA, Cat#302008); for staining memory cells anti-KLRG1 FITC (BioLegend, USA, Cat#138410), anti-CD27 PE (BD Biosciences, USA, Cat#555441); for intracellular staining anti-IL-32α (R&D Systems, USA, Cat#IC30402A) and anti-IFN-γ APC (BioLegend, USA, Cat#502512) antibodies were used. Magnetic beads conjugated to anti -CD56 (Miltenyi Biotec, Germany, Cat# 120-000-307) and -CD14 antibodies (Miltenyi Biotec, Germany, Cat# 120-000-305) were used for positive selection of NK cells and monocytes respectively.
For in vitro stimulation assays, we used ESAT-6 (BEI Resources, USA, Cat#NR-50711) and CFP-10 (BEI Resources, USA, Cat#NR-50712) peptide pools consisting of 21 and 22 individual peptides belonging to 6-kDa ESAT-6 and 10-kDa CFP-10 respectively. γ-irradiated Mtb H37Rv whole cells were used for some experiments (BEI resources, USA, Cat#49098).