Apc conjugated anti cd14 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.

All relevant cells were harvested and rinsed utilizing FACS buffer (0.05% sodium azide and 0.5% bovine serum albumin in phosphate‐buffered saline (PBS; Thermo Fisher Scientific). For excluding dead cells, cells were firstly stained with zombie aqua reagent (#423101; Biolegend) in PBS for 10 min at 20°C in the dark. Relevant cells were stained utilizing the APC conjugated anti‐CD14 antibody (#367107; Biolegend), fluorescein isothiocyanate‐conjugated CD33 antibody (#303304; Biolegend), APC conjugated anti‐Gr‐1 antibody (#108424; Biolegend), Brilliant Violet 421 conjugated anti‐Ly6G antibody (#127628; Biolegend), Alexa Fluor® 488 conjugated anti‐CD8 antibody (#100723; Biolegend), APC‐conjugated CD4 antibody (#100412; Biolegend) in FACS buffer as mentioned for at least 30 min at 4°C in the dark. MFI of ROS was detected using CellROX™ Green Flow Cytometry Assay Kit (C10492; Thermo Fisher Scientific). Flow cytometry was applied on the LSRFortessa X‐20 (BD Biosciences). All data were analyzed utilizing FlowJo v10.1 (Treestar).