Fixable yellow dead cell stain

For titration experiments, cells were stained with anti-human CCR7 IgM (BD Biosciences) and fixable yellow dead cell stain (Invitrogen) at 37 °C for 30 min, washed, and incubated with anti-human CD3 BV650 (Biolegend), CD4 APC (eBioscience), CD45RO ECD (Beckman Coulter), and anti-IgM PE (Invitrogen) at 4 °C for 30 min. For coinfection experiments, cells were stained with anti-human CCR7 IgM (Becton Dickinson) and fixable yellow dead cell stain (Invitrogen) at 37 °C for 30 min, washed, and incubated with anti-human CD3 BV650 (Biolegend), CD4 APC (eBioscience), CD45RA APC-Cy7 (Biolegend), and anti-IgM PE (Invitrogen) at 4 °C for 30 min. Upon completion of antibody incubations, cells were washed with PBS and resuspended in 1 % paraformaldehyde in PBS/BSA prior to analysis using a BD LSRII flow cytometer. At least 50,000 events were collected per sample. FlowJo version 9.6 (Tree Star, Inc.) was used for analysis.

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.