Cd103 percp cy5

Two panels of fluorochrome-conjugated antibodies (BioLegend, San Diego, CA, USA) were used to identify the innate immunity cell populations and to analyze the activation marker expression: (1) CD11b-PE/Cy7, CD11c-PE, MHCII-Alexa488, CD103-PerCP-Cy5.5, CD45-APC/Cy7, CD64-BV421, CD24-BV510; (2) CD45-APC/Cy7, MHCII-Alexa488, Ly6G-PerCP-Cy5.5, CD86-BV421, CD83-BV510. T-cells staining was performed using the fluorochrome-conjugated antibody set containing CD4-PerCP-Cy5.5, CD8-PE/Cy7, CD62L-APC/Cy7, and CD44-BV421 (BioLegend, San Diego, CA, USA). Intracellular production of cytokines was assessed using antibodies against IFNγ-FITC, IL2-PE, and TNFα-BV510 (BioLegend, San Diego, CA, USA). Staining for the detection of intracellular markers was performed using the Fixation and Permeabilization Solution reagent kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Zombie Red viability marker (BioLegend, San Diego, CA, USA) was used to identify the dead cells. True Stain reagent (BioLegend, San Diego, CA, USA), containing antibodies to CD16/CD32, was added during the surface markers staining to block non-specific antibody binding. Data were collected on a Cytoflex flow cytometer (Beckman Coulter, Bray, CA, USA). The results were analyzed using the Kaluza Analysis 2.2 program (Beckman Coulter, Bray, CA, USA).

Fc receptors on isolated cells were blocked with a 1:300 dilution of anti-CD16/32 (eBioscience) for 30 min at 4°C, washed with PBS-BSA 0.2%, and labeled with a 1:400 dilution of Ly-6C (APC-Cy7; BD Bioscience), CD11c (PE-Cy7; BD Bioscience), B220 (V500; BD Bioscience), CD103 (PerCP-Cy5.5; BioLegend), a 1:1600 dilution of MHCII (APC; BioLegend), and a 1:4000 dilution of CD11b (PE; BD Bioscience) for 30 min at 4°C. Cells were washed twice in PBS-BSA 0.5% and fixed in 2% PFA diluted in PBS-BSA 0.2% for 15 min at 4°C. Cells were washed, resuspended in PBS-BSA 0.2%, and analyzed by flow cytometry. Samples were collected using a BD FACS Aria III with FACSDiva software and post-acquisition analyses were performed using FlowJo.

Hepatic mononuclear cells (HMNCs) and splenocytes were isolated as previously described [18 (link)]. Single-cell suspensions of HMNCs and splenocytes were labelled with fluorescent conjugated antibodies against mouse CD4-FITC, CD25-PE (eBioscience), CD62L-PE Cy7, CTLA-4-PE (BD Pharmingen), as well as CD103-PerCP-Cy5.5 (Biolegend). Nonspecific binding was blocked with staining buffer supplemented with 2% mouse serum and anti-CD16/anti-CD32. For intracellular staining of Foxp3, mouse regulatory T cell staining Kit (eBioscience) was used. Cells were labeled with surface antibody, fixed and permeabilized with freshly prepared Fixation/Permeabilization working solution according to the manufacturer’s instructions. After permeabilization, cells were further labeled with anti-mouse foxp3 (clone FJK-16s) (eBioscience). After incubation and resuspension, HMNCs and splenocytes were evaluated by flow cytometry, and the data were analyzed using Cell Quest software (Becton Dickinson).

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.

Single cells were incubated with Human TruStain FcX (BioLegend) to block Fc receptors. These were then stained with Ghost Dye™ Red 780 Viability Dye (Cell Signaling Technology) to identify dead cells and the following fluorochrome-conjugated anti-mouse monoclonal antibodies (mAbs): CD103-PerCP/Cy5.5 (Ber-ACT8), CTLA-4- PerCP/Cy5.5 (BNI3), Granzyme B-FITC (QA16A02), PD-1-PE (EH12.2H7), CD27- PE/Cy7 (LG.3A10), CD3- PE/Cy7 (HIT3a), CD8a-AF700 (RPA-T8), CD4-APC (OKT4), CD127-BV711 (A019D5), Ki-67-BV605 (Ki-67), CD45RA-BV605 (HI100), CCR7-BV510 (G043H7), CD69-BV421 (FN50), and IFNγ-BV421 (4S.B3) (all from BioLegend). Data were acquired on a LSRFortessa X-20 (BD) and analyzed with FlowJo software.