Clone m5e2

Antibodies to the surface epitopes CD14 (clone HCD14, Pacific Blue-conjugated; and clone M5E2, Brilliant Violet 510-conjugated), CD16 (clone 3G8, PE-conjugated), TLR2 (clone TL2.1, PE-conjugated) and TLR4 (clone HTA125, Brilliant Violet 421-conjugated) as well as isotype controls (clone MOPC-173, clone MOPC-21, clone RTK2071) were purchased from BioLegend. For intracellular cytokine staining, antibodies to TNF-α (clone MAb11, PerCP/Cy5-5-conjugated), IL-1β (clone H1b-98, Alexa Fluor 647-conjugated), IL-8 (clone E8N1, Alexa Fluor 488-conjugated) and IL-10 (clone JES3-9D7, PE/Cy7-conjugated) were also obtained from BioLegend. Fixable viability stain (eFluor® 780, APC-H7-conjugated) was purchased from eBioScience (San Diego, CA).

100,000 cells per polystyrene FACS tube (BD Pharmingen™) were treated according to the Monocyte treatment section with slight change. After 1 h of treatment with sera, Brefeldin A (Invitrogen) was added to each tube to 1x concentration and monocytes were incubated for further 2 h at 37°C and 5% CO₂. Subsequently, monocytes were incubated with Phycoerythrin-conjugated anti-human CD14 antibody (2 μL; Clone M5E2; BioLegend) and fixable yellow dead cell stain (2 μL; Invitrogen, Carlsbad, California, US). After 30 min at RT, cells were washed with FACS buffer and centrifuged at 400 g for 5 min. Supernatant was removed and monocytes were fixed with Fix and Perm Medium A at room temperature for 15 min. After further washing step, cells were permeabilized with Fix and Perm Medium B (both Invitrogen, Carlsbad, California, US) and incubated with PerCP/Cyanine5.5-conjugated anti-human TGF-β1 antibody (2 μL; Clone TW4-2F8; BioLegend) at room temperature for 30 min. Following the washing step, monocytes were resuspended in 50 μL FACS buffer and analyzed using BD FACS Canto 2™ and FACD DIVA™ software (BD). Monocytes were gated by the corresponding forward and side scatter scan and as shown in Figures 5A,B. The percentage of TGF-β1 expression of viable CD14⁺ monocytes was analyzed.

It has been observed that monocytes spontaneously and rapidly aggregate in vitro at 4°C, called cold-aggregation purification procedure [5 ]. Therefore, monocytes were segregated from lymphocytes by resuspending the PBMCs in RPMI-1640 medium (Sigma Immunochemicals, USA) supplemented with 10% fetal bovine serum (FBS, Cultilab, Brazil) and incubating for 30 min at 4°C with continuous agitation. Monocytes then spontaneously sedimented [5 ], and two successive rounds of cold-aggregation were subsequently performed. We determined cell viability by trypan blue dye. The monocyte population was determined by flow cytometry based on forward and side scatter (FSC vs. SSC) gating, as well as on the expression or lack of the surface markers anti-hCD14-FITC (Dilution 1:100; Clone M5E2; BioLegend, USA; cat 982502) and anti-hCD3-Alexa 647 (Dilution 1:100; Clone OKT3; eBiosciences, USA; cat 51-0037-73).