Pe conjugated anti human cd163

In order to obtain single cell suspension, the adherent cells were digested by 0.25% trypsin with 0.02% EDTA (Genom, Shanghai, China), and the tissues were digested with Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12) containing 1.0 mg/ml collagenase IV (Sigma-Aldrich, MO, USA) and 150 U/ml DNase I (Sigma-Aldrich, MO, USA). The expression of cell-surface and intracellular molecules was detected by flow cytometry. For cell-surface molecules, the cells were incubated with following antibodies: fluorescein isothiocyanate (FITC)-conjugated anti-mouse F4/80; PerCP/Cyanine5.5 anti-mouse CD11c; Brilliant Violet (BV) 421-conjugated anti-mouse CD86; (phycoerythrin (PE)-conjugated anti-human CD163; allophycocyanin (APC)-conjugated anti-human CD206; PE/Cyanine7 anti-human CD86; PerCP/Cyanine5.5 anti-human CD209 (Biolegend, CA, USA). For intracellular molecules tests, the cells were fixed and permeabilized with Fix/Perm Kit (Biolegend, CA, USA) and then incubated with antibodies: APC-eFluor 780-conjugated anti-mouse iNOS (Ebioscience, CA, USA); PE-conjugated anti-mouse Arg1 (Invitrogen, MA, USA)). A minimum of 10,000 events were collected by a BD or Beckman flow cytometer and analyzed with FlowJo or CytExpert software.

PBMC or iPS-MLs were incubated with FcR blocking antibodies (eBioscience, San Diego, CA, USA) on ice for 15 min, and subsequently labeled with specific antibodies at 4°C. The following antibodies were used: phycoerythrin (PE)-conjugated anti-human CD11b (Clone: ICRF44; eBioscience), fluoresceinisothiocyanate (FITC)-conjugated anti-human CD45 (Clone: HI30; BD Biosciences, Bedford, MA, USA), PE-conjugated anti-human CD33 (Clone: WM53; BD Biosciences), FITC-conjugated anti-human CD14 (Clone: 61D3; eBioscience), PE-conjugated anti-human CD16 (Clone: eBioCB16; eBioscience), PE-conjugated anti-human CD163 (Clone: RM3/1; BioLegend, San Diego, CA, USA), and PerCP-conjugated anti-human CD206 (Clone: 15–2; BioLegend). FITC-, PE-, and PerCP/Cy5.5-conjugted mouse IgG1 κ (Clones: P3.6.2.8.1 and MOCP-21) were used as isotype-matched controls. Stained cells were analyzed using FACS Calibur (BD Biosciences).