Cd80 pe

For detection of branched complex glycoconjugates, cells were stimulated with 50 ng/mL LPS for 24 h and 48 h hours or left untreated. Cells were washed in ice-cold 1% bovine serum/PBS and incubated with 2 µg/200,000 cells Alexa488-coupled PHA-L lectin (Invitrogen). For the detection of differentially expressed glycoproteins after LPS treatment, cells were stimulated for 4 h, 24 h, or 48 h with or without LPS and labelled with specific fluorescent-dye conjugated antibodies CD54-APC, Immunotools; CD14-FITC, CD169-APC, CD86-PerCP-Cy5 CD127-APC, CD274-APC, CLEC12A-PE, all eBiosciences; CD319-Alexa647, BD Biosciences; CD80-PE, Miltenyi Biotec). After washing, flow cytometry was performed with an Attune Acoustic Focusing Cytometer Attune (Applied Biosystems) or FACS-ARIA II (BD Biosciences). For visualization, FCS-files were loaded into FlowJo. Expression of glycoprotein marker proteins was analyzed by gating on live CD14⁺ cells.

Cells were detached mechanically using cell scrapers and resuspended in staining buffer (PBS containing 2 mM of EDTA and 4% FBS), followed by incubation with human FcR Blocking Reagent (Miltenyi Biotec). Cells were counted and 1 × 10⁵ cells were stained with fluorochrom-conjugated antibodies against CD163-FITC (BioLegend, California, USA), CD86-APC (Miltenyi Biotec), CD83-APC (Miltenyi Biotec) and CD80-PE (Miltenyi Biotec) for 20 min on ice protected from light. Cells were washed twice with staining buffer and fixed with PBS containing 4% paraformaldehyde (Electron Microscopy Sciences, PA, USA). 8000 events were acquired using Gallios Flow Cytometer (Beckman Coulter, CA, USA) and analyzed using the Kaluza software (Backman Coulter).

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.