Anti cd86 pe

FACS analysis was performed according to previous reports [64 (link)]. Briefly, staining of cells for flow cytometry was performed in suspension cell from kidney tissue using between 1 × 10⁵ and 1 × 10⁶ cells per tube. For phospho-flow staining, kidney cells were resuspended in ice-cold PBS and immediately an equal volume of prewarmed BioLegend fixation buffer (catalog 420801, San Diego, CA, USA) was added and samples were incubated at 37 °C for 15 min. The BioLegend intracellular staining with True-Phos Perm Buffer (catalog 425401, San Diego, CA, USA) protocol was followed and all washes were performed with BioLegend Cell Staining Buffer (catalog 425401, San Diego, CA, USA). BioLegend Trustain FcX for mouse (clone 93, catalog 101320, San Diego, CA, USA) were used to block samples before staining. Phospho-flow experiments were collected on a BD LSRII and kidney homogenate analyses were collected using a BD FACS Canto RUO. FlowJo software (BD) was used for analysis of flow cytometry data.
The following antibodies were used: anti-CD86-PE (105007; Biolegend, San Diego, CA, USA), anti-F4/80-FITC (101205; Biolegend, San Diego, CA, USA), anti-CD206-PE (141720; Biolegend, San Diego, CA, USA), and anti-galectin-3-PECy7 (125418; Biolegend, San Diego, CA, USA).

After the intervention, Mφs were collected and incubated with anti-F4/80-APC/Cy7 (BioLegend), anti-CD11b-FITC (BioLegend), anti-CD86-PE (BioLegend), and anti-CD206-APC (BioLegend) antibodies. Flow cytometry was performed on an FACSCalibur (BD Biosciences) and analyzed using FlowJo software. The gating strategy for M2 polarization was as follows: the CD11b⁺- and F4/80⁺-populations were gated first, and then the CD11b⁺ F4/80⁺ population was further gated based on CD206 intensity.

Murine leukocytes were stained with fluorescent monoclonal antibodies against F4/80 (labeled with PE-Cy7,), GR1 (labeled with APC,), CD11b (labeled with V500), CD206 (labeled with APC), CD86 (labeled with PE), anti-rabbit secondary (labeled with alexa 488 and with alexa 405). Stained cells were acquired in a NovoCyte (ACEA Biosciences, San Diego, CA, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA). Macrophage populations were defined according to F4/80, GR1, and CD11b expression, as previously described (24 (link), 28 (link)). For human cell analyses, macrophages were labeled with 1.25 μg/mL anti-HLA_DR/DP/DQ-FITC (BD Pharmingen) and 1 μg/mL anti-CD86-PE (BioLegend), at 4°C for 30 min. Cells were acquired on a LSRFortessa cytometer (BD Biosciences).

The monoclonal antibodies for the characterization of DC and lymphocytes by flow cytometry were the following: anti-CD3 FITC, anti-CD4 APC, anti-CD8 PE, anti-CD137-biotin, anti-CD11c APC, anti-CD40-PE, anti-CD86-PE, anti-CD80-PE, anti-F4/80-Cy5 and anti-Ia/Ie-FITC from Biolegend (San Diego, CA, USA). The murine melanoma cell line B16-F10 with haplotype H-2Kb was purchased from The American Type Culture Collection, USA. RPMI-1640 culture medium was purchased from Biowest (Nuaille, France). Ge from bovine skin with a protein percentage of 78% and a water content of 8% was used. HA sodium salt from Streptococcus equi, both from SIGMA (St Louis, MO, USA) with a solubility of 5 mg/mL and an MW of ~1.5–1.8 × 10⁶ Da was used.

At 24 h post-stimulation, supernatant and MoDCs were collected for analysis. Antiviral cytokines levels in the supernatant were measured using cytometric bead array [LEGENDplex™ Human Anti-Virus Response Panel (13-plex), BioLegend, San Diego, CA, USA].
Phenotypic maturation of MoDCs was analyzed by flow cytometry using anti-CD14 PB, anti-CD86 PE, anti-MHC II FITC, anti-CD83 FITC, and anti-CD155 APC (Biolegend, San Diego, CA, USA).