Cd86 pe vio770

For DC analysis in the skin and sdLNs, cells were stained with different combinations of the following antibodies: MHC-II-Vioblue, CD11c-PerCPVio700, CD11b-VioGreen, CD11b-PerCPVio700, XCR1-Viobright-FITC, EPCAM-PE, CD86-PE-Vio770, CD86-APC-Vio770, PD-L2-PE-Vio770 (all from Miltenyi Biotec), CD11c-APC-Cy7 (BD Biosciences), XCR1-BV510 (Biolegend). Dead cells were excluded from analysis using Zombie dye (Biolegend) of appropriate color depending of the antibodies used.
For Tregs analysis, cells were stained with combinations of the following antibodies: anti-mouse CD4-FITC (Miltenyi), CD25-APC-Cy7, CD62L-PE-Cy7 (all from BD Biosciences, Le Pont de Claix, France), Latency-associated peptide (LAP)-PE and Foxp3-APC (from e-Bioscience, Paris, France), or control isotypes. Intracellular staining was performed after fixation and permeabilization, using Foxp3 Perm Kit (e-Bioscience, Paris, France). Dead cells were excluded from analysis using Zombie dye aqua (Biolegend).
Flow cytometry was performed on a MACSquant cytometer and analyzed using FlowJo software.

All data were acquired using a Cytomics FC 500 flow cytometer operating on CXP software equipped with a dual 488 nm/635 nm (blue/red) laser (Beckman Coulter, Brea, CA, USA). Intracellular staining and staining of cell surface antigens were performed simultaneously using the MACS Inside Stain Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, Inside Fix buffer was added to 1 × 10⁵ cells and then resuspended in PBS. The solution was incubated at room temperature and pressure for twenty minutes, after which the cells were washed twice with PBS via centrifugation for five minutes at 300 g. The supernatant was discarded, and the pellet was resuspended in 100 µL of Inside Perm buffer; cells were stained with fluorochrome-conjugated antibodies at appropriate dilutions. Cells were incubated for fifteen minutes at room temperature and then washed with Inside Perm buffer. The supernatant was discarded, the pellet was resuspended in 400 µL of PBS, then flow cytometric analysis was performed. Antibodies against CD11b-VioBright FITC, CD86-PE-Vio770, and CD68-FITC were obtained from Miltenyi Biotec, (Bergisch Gladbach, Germany), whilst antibodies against CD206-PE and CD163-PE were obtained from eBioscience (San Diego, CA, USA).

Blood samples from the baseline visit were processed within 4 hours for analysis by flow cytometry. For lymphocyte phenotyping, ethylenediaminetetraacetic acid whole blood was stained for CD3, CD4, CD8, CD45, CD16, CD56, and CD19, as previously described (30 (link)). For B-cell phenotyping, PBMCs were isolated from lithium heparin whole blood by Ficoll gradient density centrifugation. One million PBMCs were incubated with the following antibodies: CD19-VioGreen, anti-IgD-VioBlue, CD24-PerCP-Vio700, CD38-FITC, CD27-APC, CD86-PE-Vio770, CD21-APC-Vio770, and anti-IgM-PE (Miltenyi Biotec, Bergisch Gladbach, Germany). Samples were measured using a FACSLyric flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were analyzed using the FACSSuite (BD Biosciences). The gating strategy is shown in Supplemental Figure 1.