Percp anti human cd16 antibody

The experimental protocol was conducted as described by Nakaira-Takahagi et al., with some adaptations [34 (link)]. PMNs were initially treated or not with monoclonal antibodies anti-human TLR2 (0.8 μg/10⁵ cells) (BioLegend, San Diego, CA, USA) and mouse anti-human TLR4 (2 μg/10⁵ cells) (BD Pharmingen™, Franklin Lakes, NJ, USA), individually or in combination, for 2 h in a 5% CO₂ atmosphere at 37°C. After the receptor blockade, cells were incubated in the presence of gp43 (20 ng/ml) or in its absence (control culture) for 4 hours at the same conditions described above. Supernatants were collected, centrifuged, and stored at -20°C for measuring IL-6, IL-10, IL-12, IFN-γ, PGE2, and LTB4 levels. PMNs were then incubated with PerCP anti-human CD16 antibody, FITC anti-human CD282 (TLR2) antibody (both antibodies from BioLegend, San Diego, CA, USA), and PE mouse anti-human TLR4 antibody (BD Pharmingen™, Franklin Lakes, NJ, USA) for 20 minutes at 4°C in the dark. Receptor expression was determined by FACSCanto II flow cytometry (BD, San Diego, CA, USA) using the FACSDiva software. The standard acquisition was set to 25,000 events, and cells were gated based on size (forward scattered (FSC)), granularity (side scattered (SSC)), and fluorescence parameters (PerCP-CD16+ and/or FITC-TLR2+ and/or PE-TLR4+). Data were analyzed with FLOWJo software.

For the degranulation assay, K562 cells were co-cultured with vehicle-treated or Verbenalin-treated NK cells in the presence of Brilliant Violet 421™ anti-human CD107a (LAMP1) antibody (Biolegend, San Diego, CA, US) and GolgiStop^TM (BD Biosciences). The incubation was carried out at 37 °C with 5% CO₂ for 4 h. The cells were then stained with PerCP anti-human CD16 antibody (Biolegend) and APC mouse anti-human CD56 antibody (BD Biosciences) to define NK cells. Data were obtained with a FACSVerse™ flow cytometer (BD Biosciences) and analyzed with FlowJo v10 (FLOWJO, LLC).