Cd163 pe

Immunofluorescence staining of cell surface antigens in MSC was performed by incubating cells for 30 min at 4 °C in the dark with mouse anti-human leukocyte antigen (HLA)-DR, DP, DQ (HLA class II)-FITC, HLA-ABC (HLA class I)-APC, CD34-FITC, CD44-FITC, CD105-PE, CD29-APC, and CD45-APC Abs (all from BD Biosciences, San Jose, CA,USA). Phenotypic characterization of macrophages generated by incubation with GM-CSF or M-CSF was assessed by staining with CD163-PE, CD197 (CCR7)-FITC, and CD80-APC (all from Miltenyi Biotec, Bergisch-Gladbach, Germany). Cells incubated in the absence of antibodies were used as controls. After incubation, cells were washed three times with PBS, fixed with 1% (w/v) formaldehyde in PBS, and analyzed by flow cytometry using a FACSCalibur analyzer and CellQuest software (both from BD Biosciences).

The primary antibodies used were CD14 VioBlue (BD Biosciences, Heidelberg, Germany; catalog number 558121), CD16 FITC (Miltenyi Biotec, Bergisch Gladback, Germany; catalog number 130‐113‐392), CD80 FITC (BioLegend, San Diego, CA, USA; catalog number 305205), CD86 PE (BioLegend, San Diego, CA, USA; catalog number 305405), CD163 PE (Miltenyi Biotec, Bergisch Gladback, Germany; catalog number 130‐097628), CD169 APC (Miltenyi Biotec, Bergisch Gladback, Germany; catalog number 130‐098‐643), CD206 APC (BD Biosciences, Germany; catalog number 550889) and CD209 VioBlue (BioLegend, San Diego, CA, USA; catalog number 330102). Antibodies were used as per manufacturer's instructions and incubated for 30 min at 4°C. Data were acquired using a MACSQuant flow cytometer (Miltenyi Biotec, Bergisch Gladback, Germany) and analyzed using FlowJo ™ version 10.7 (BD Biosciences, San Jose, CA, USA).

Receptor expression (CD32, CD64, CD80, TLR2, CD163, CD36 and CD206) was evaluated by flow cytometry at the membrane of differentiated human macrophages after 3 days of co-culture with opsonized SKOV3-R2⁺ tumor cells. Receptors were detected using CD32-PE-Vio770, CD64-PerCP-Vio700, CD80-PE, CD282 (TLR2)-APC, CD163-PE, CD36-PE and CD206-APC (Miltenyi, Bergisch Gladbach, Germany) and were compared with an appropriate isotype control.
A population of 10,000 cells was analyzed for each data point. Dead cells (positive cells) were removed from the analysis after labeling with Viobility Fixable Dye (Miltenyi, Bergisch Gladbach, Germany). Analyses were gated on CD14 or Cd11b positive cells. All analyses were performed using a BD Fortessa flow cytometer with the Diva software. The gating strategies are presented in the Figure S6.