Pe conjugated anti cd16 antibody

2ml heparin-treated blood was used to isolate the human peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation Ficoll Paque (GE Healthcare, Piscataway, NJ, USA). FITC-conjugated anti-ALK4 antibody (R&D), PE-conjugated anti-CD16 antibody (Biolegend), and APC-conjugated anti-CD14 antibody (Biolegend) were used for flow cytometry. After the PBMCs were incubated with antibodies, washed, and fixed, the samples were analyzed by Flow software (CytExpert) and flow cytometry (CytoFLEX, Beckman Coulter, Inc. Brea, CA, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated from 2 ml heparin-treated blood by density gradient centrifugation using Ficoll Paque (GE Healthcare, Piscataway, NJ, USA). After blocking the Fcγ receptors, PE-conjugated anti-CD16 antibody (Biolegend) and APC-conjugated anti-CD14 antibody (Biolegend) were used for flow cytometry. After the PBMCs were incubated with antibodies, washed and fixed, the samples were analyzed by flow cytometry (BD Accuri C6 instrument, BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software (Tree Star).
M0 and M1 RAW264.7 macrophages were incubated with PE-conjugated anti-KCa3.1 antibody (Santa Cruz) to analyze the expression of KCa3.1, and were incubated with Fluo-3AM (Sigma) to detect intracellular Ca²⁺ by flow cytometry.