Fitc anti cd206 antibody

THP-1 cells were seeded in 60 mm dishes, differentiated and treated as indicated. Cells were washed with cold PBS; detached with versine; pelleted; resuspended in a total of 100 µL of cold PBS containing 5 µL of PE anti-CD80 antibody (number 557227) (Becton Dickinson Italia, MI, Italy) or FITC anti-CD206 antibody (number 321103) (BioLegend, San Diego, CA, USA); and incubated 15 min at room temperature in the dark. After incubation, cells were washed with 1 × PBS and centrifuged at 500 × g for 5 min and then re-suspended in 500 μL of 1 × PBS. Cells were analyzed by FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) and the data acquired using CellQuest software (version 3.3). Unstained cells were used to determine the background autofluorescence to set the negative population allowing cells stained with anti-CD80 (or anti-CD206) antibody to be visualized.

THP-1 cells were seeded in 60 mm dishes, differentiated as previously described and untreated or treated with C-EV or Lep-EV for 48 h before staining. Cells were washed with cold PBS; detached with versene and centrifuged, and the obtained pellet was resuspended in a total of 100 µL of cold PBS containing 5 µL of PE anti-CD80 antibody (# 557227; Becton Dickinson, MI, Italy) or FITC anti-CD206 antibody (# 321103; BioLegend, San Diego, CA, USA). After incubation (30 min at 4 °C), cells were washed with 1× PBS and centrifuged at 500× g for 5 min and then re-suspended in of 1× PBS and analyzed by flow cytometry (CytoFLEX Beckman, Beckman Coulter, Milan, Italy). Data analysis was performed using CytExpert Beckman Coulter software (Beckman Coulter, Milan, Italy). Isotype matched negative control antibodies were used as negative control sample.