Anti mouse cd11c apc

WT C57 BL/6 mice bone marrow DCs were prepared as described above and then respectively stimulated with 5 μg/ml of rTs-Hsp70, 50 ng/ml of lipopolysaccharide (LPS, a agonist of TLR4, InvivoGen, USA), 200 ng/ml of Pam₃CysSerLys₄ (Pam₃CSK₄, a agonist of TLR2, InvivoGen, USA) on day 5 for 48 h. Control cells were added with 20 μl phosphate-buffered saline (PBS) or 5 μg/ml of bull serum albumin (BSA, Thermo, USA). The stimulated DCs were collected and washed with 3 ml PBS once. The collected DCs were firstly pre-incubated with Fc Blocker (Anti-Mouse CD16/CD32, BD Biosciences, USA) for 30 min to reduce non-specific binding of labelled antibodies, then stained with anti-mouse CD11c APC (eBioscience, USA), anti-mouse CD282 (TLR2) FITC (eBioscience, USA) or anti-mouse CD284 (TLR4) PE (eBioscience, USA) on ice for 30 min, respectively. The percentage of TLR2 and TLR4 expressing cells (CD11c-APC and TLR2-FITC/TLR4-PE double positive) was analyzed by Flow Cytometry (BD Biosciences, USA). Doublets were excluded from all analysis.

Handpicked islets were pooled in 24-well plates for 24 h in RPMI supplemented with 10% FCS, penicillin–streptomycin, and 5.10⁻⁵ M β-mercaptoethanol allowing extrusion of infiltrating lymphocytes (25 (link)). Infiltrating lymphocytes were recovered. Cells recovered were used for the analysis of T cells and antigen-presenting cells. T-cell analysis was performed using the Foxp3 intra-staining buffer set according to the recommendation of the manufacturer (eBioscience) with the following combination: anti-mouse CD45-efluo450, anti-mouse CD3ϵ-AlexaFluo700, anti-mouse B220-HorizonViolet500, anti-mouse CD8α-Percp-Cy5.5, anti-mouse CD4-PE, anti-mouse Foxp3-APC, and anti-mouse CD25-BrillantViolet711 mAbs (eBioscience). β Cells and dendritic cells were analyzed with anti-mouse CD45-AlexaFluo700, anti-mouse TCRβ-APC-Cy7, anti-mouse B220-HorizonViolet500, anti-mouse CD11b-efluor450, anti-mouse CD11c-APC, anti-mouse CD8α-BrillantViolet605, and anti-mouse CD4-PE mAbs (eBioscience). Acquisitions were performed with a BD LSR-Fortessa flow cytometer and analyzed using FlowJo 10.7.1 software.

Pulmonary conventional DC subsets were analyzed by FACS from Sema3e^-/- or WT mice 3 days after intranasal exposure with a single high dose of HDM [3 (link)]. Briefly, lungs were removed from mice and enzymatically digested using 1 mg/ml collagenase IV (Worthington Biochemical Corporation, Lakewood, NJ) and 0.5 mg/ml DNase from bovine pancreas in RPMI 1640 medium. After Fc blocking, DCs were stained by anti-mouse CD11c-APC (Clone: N418, eBioscience), MHCII eFluor® 450 (Clone: M5/114.15.2, eBioscience), CD11b-PE-Cy7 (Clone: M1/70, BioLegend), and CD103-PerCP-Cy5.5 (Clone: 2E7, BioLegend). Anti-mouse PD-L2-PE (Clone: TY25, BioLegend) and IRF-4 (Clone: IRF4.3E4, BioLegend) antibodies were separately added to the tubes followed by acquisition of the samples using a BD FACS Canto-II (BD, San Diego, CA) and analyzed using FlowJo V10.7.

After incubation with CPP-conjugated molecules, the cells were resuspended in 100 μl antibody mixture diluted in PBS and incubated at 4°C for 15 min. To discriminate each cell subset from the mixed cell population, different antibody combinations were used. For cell type analysis, anti-mouse CD4-PerCP-Cy5.5 (BioLegend, Clone:RM4-5), anti-mouse CD19-PE-Cy7 (BioLegend, Clone:6D5), anti-mouse NK1.1-APC (BioLegend, Clone:PK136), anti-mouse CD11b-PE-Cy7 (eBioscience, Clone:H57-597), anti-mouse CD11c-APC (eBioscience, Clone:N418), anti-mouse I-A/I-E-APC-Cy7 (BioLegend, Clone:M5/114.15.2), and anti-mouse F4/80-PerCP-Cy5.5 (BioLegend, Clone:BM8) were used. Also, anti-mouse CD16/32 (BioLegend, Clone:93) was used for blocking the Fc receptor. For activated T cell analysis, anti-mouse CD69-FITC (BioLegend, Clone:H1.2F3), anti-mouse CD4-APC (eBioscience, Clone:GK1.5), and anti-mouse CD8-PerCP-Cy5.5 (eBioscience, Clone:53–6.7) were used. For activated B cell analysis, anti-mouse CD86-PE-Cy5 (BioLegend, Clone:GL-1), anti-mouse IgG2a, kappa-isotype (BioLegend, Clone:RTK2758), and anti-mouse CD19-PE (BioLegend, Clone:6D5) were used.

Spleen immune cells were isolated from 129 mice (n = 6) and Dok-3^−/− mice (n = 6). Mice spleens were isolated from the body and grinded by the copper grid. The immune cells of spleen were obtained after the 10 min incubation with red blood cell lysis buffer. The cells were stained with anti-mouse Tim-3-PE (ebioscience), anti-mouse CD3-APC (ebioscience) or anti-mouse CD3-FITC (ebioscience) which was used in DC cells marking, anti-mouse CD4-FITC (ebioscience), anti-mouse CD8-FITC (ebioscience), anti-mouse CD11b-Pe-cy-7 (ebioscience), anti-mouse NK1.1-PerCP-5.5 (ebioscience), anti-mouse CD11c-APC (ebioscience), anti-mouse Gr-1-FICT (ebioscience) for 30 min. At least 10,000 cells were analyzed by a FACSAriaII. Cells were gated based on their forward and side scatter properties. The cells’ gated strategy is shown in Additional file 1: Figure S1.

Bone marrow-derived macrophages were transduced with a control vector (pMX-FLAG-IRES-EGFP) or the constitutively active form of STAT5A (pMX-FLAG-IRES-EGFP-STAT5A1*6), differentiated to osteoclasts in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL), and collected. The collected cells were stained with PE-anti-mouse CD80 (BD Pharmingen^TM, San Diego, CA, USA), PE-anti-mouse CD 86 (BD Pharmingen^TM), PE-anti-mouse MHC class II (eBioscience, San Diego, CA, USA), and APC-anti-mouse CD11c (eBioscience) for 20 min at 4 °C. Stained cells were analyzed using a Navios flow cytometer with Kaluza software (Beckman Coulter, Brea, CA, USA).