Cd11b viogreen

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.

10 weeks old female C57BL/6J mice (n = 3 animals per group) were s.c. immunized at the inguinal site at both right and left flanks, with fluorescent Cy5‐labeled plain (empty) or Adpgk‐loaded NP (100 µg of Adpgk/20 µg of CpG/40 µg of Poly(I:C) per mouse; 100 µL of NP per side). PBS‐treated mice were used as negative controls. Inguinal LN were harvested 14 h post‐immunization. A single cell suspension was stained with fluorochrome‐labeled anti‐mouse antibodies against CD11b‐VioGreen (Miltenyi Biotec, Cat.# 130‐113‐811, clone: REA592, 1:50), CD11c‐FITC (Miltenyi Biotec, Cat.# 130‐110‐837, clone: REA754, 1:50), MHC Class II (I‐Ab)‐VioBlue (Miltenyi Biotec, Cat.# 130‐112‐237, clone: REA813, 1:50), CD80‐PE‐Vio 770 (Miltenyi Biotec, Cat.# 130‐116‐462, clone: REA983, 1:50), and CD86‐PE (Miltenyi Biotec, Cat.# 130‐122‐129, clone: REA1190, 1:50), for 15 min at 4 °C. Samples were analyzed using a LSR Fortessa cytometer (BD Biosciences) and FlowJo software version 7.6.5 for Microsoft (TreeStar).

Cells were harvested after differentiation and washed thrice in autoMACS running buffer (Miltenyi Biotec, #130-091-221, Gladbach, Germany). Cells were transferred to 96-well plates with 2 × 10⁵ cells for each antibody panel. Cells were stained with the following antibodies: diluted 1:50, REA Control (S)-VioGreen (Miltenyi Biotec, #130-113-444), REA Control (S)-PE (Miltenyi Biotec, #130-113-438), REA Control (S)-APC (Miltenyi Biotec, #130-113-434); REA Control (S)-PE-Vio770, (Miltenyi Biotec, #130-113-440); HLA-DR-VioGreen (Miltenyi Biotec, #130-111-795), CD54-APC (Miltenyi Biotec, #130-121-342); CXCR4-PE-Vio770 (Miltenyi Biotec, #130-116-161); CD11b-VioGreen (Miltenyi Biotec, #130-110-617); CD83-PE (Miltenyi Biotec, #130-110-561); CD86-APC (Miltenyi Biotec, #130-116-161) for 10 min at 4 °C in the dark. Afterwards, cells were washed thrice with autoMACS running buffer and stained with DAPI (Sigma, #D9542), to exclude dead cells for the determination of the cell viability.