Anti il 10 pe

To evaluate the recruitment of Th1, Th2, and Tregs induced by ASCs treatment, LLN cells from OVA-induced asthmatic mice and ASC-treated asthmatic mice were cultured in anti-CD3-coated plates for 6 h. To evaluate CD4⁺CD25⁺Foxp3⁺ (Tregs) and IL-10⁺/CD4+ T-cells, cells were stained with anti-CD4-FITC (0.5 mg/mL) and anti-CD25-APC (0.2 mg/mL) following the manufacturer’s recommendations (eBiosciences, San Diego, CA). After surface staining, cells were permeabilized using the Cytofix/Cytoperm Kit (BD Biosciences). After permeabilization, cells were stained with anti-Foxp3-PE-cy7 or anti-IL-10-PE (eBiosciences).
To assess the Th1 and Th2 cell populations, LLN cells were stained with anti-CD4-FITC Ab. After surface staining, CD4+ T-cells were stained with intracellular anti-IFN-γ-PE-cy7 (eBiosciences) and anti-IL-4-PE (eBiosciences) Abs. Fluorescence was measured using a FACS CantoII cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).

anti-CD14-BV570 (301831, Biolegend), anti-CD11c-PerCP-Cy5.5 (337210), anti-HLA-DR-AlexaFlour700 (307626) all from Biolegend, anti-CD66a/c/e-QDot605 (342302, Biolegend, with in-house conjugation to QDot605, Q-22001), anti-CD16-PE-Cy5 (555408, from BD Biosciences), anti-IL-12-PE (554575), anti-TNF-α-PE-Cy7 (25-7349-82) both from eBioscience, anti-IL-10-PE (506804), anti-IL-6-APC (501112), anti-CD3-BV421 (300434), anti-CD19-BV421 (302233) and anti-CD335-BV421 (331913) all from Biolegend.
Cryopreserved, fixed white cells were washed in PBS and stained for 1 hour at 4°C with surface marker antibodies. For intracellular staining, cells were permeabilized with Perm/Wash buffer (BD Biosciences) and incubated for 1 hour at 4°C with antibodies for intracellular markers. Fixed cells were then acquired on a LSR II flow cytometer (BD Biosciences).

Flow cytometry was performed with the following antibodies: anti-CD19-Percp-Cy5.5, anti-CD19-Percp-eFluor710, anti-CD24-FITC, anti-CD38-superbright600, anti-CD38-PE, anti-Ki-67-Alexa Fluor647, anti-IL-10-PE, anti-TIM-1-PE, anti-CD4-FITC, anti-IFN-γ-PE, anti-IL-17A-APC (eBioscience, San Diego CA, USA), anti-IL-6-APC, anti-TNF-α-FITC, anti-IL-12-BV-421, and anti-CD24-Alexa Fluor647 (BD Biosciences, France). Intracellular cytokines and Ki-67 were assessed in cells treated with Permeabilization and IC Fixation Buffers (eBioscience, San Diego, CA, USA). Stained cells were analyzed with a 21-color ZE5 cell analyzer (Bio-Rad, USA). The CD19⁺CD24^hiCD38^hi B subset was sorted (FACSAria; BD Pharmingen) using anti-CD19-Percp-Cy5.5, anti-CD24-FITC, and anti-CD38-PE. The sort purity of CD19⁺CD24^hiCD38^hi B cells was routinely >90%. CD4⁺T subpopulation was sorted (FACSAria; BD Pharmingen) using anti-CD38-PE and anti-CD3-PE. The sort purity of CD4⁺T cells was routinely >95%. All flow cytometry data were analyzed with FlowjoV10 Software (Tree Star, OR, USA).

Expression of costimulatory molecules on DCs was quantified by flow cytometry using anti-CD11c-Pe/Cy7, anti-CD86-PerCP (both BioLegend), and biotinylated anti-CD40 (eBioscience; detected by streptavidin-APC/Cy7, BD Pharmingen) antibodies, with the gating strategy shown in Supplementary Figure 1. Popliteal DLN cells (10⁶/ml) from in vivo models were incubated ± 50 ng/ml PMA plus 500 ng/ml ionomycin for 1 h before addition of 10 µg/ml Brefeldin A (Sigma-Aldrich, UK) for a further 5 h at 37 °C with 5% CO₂. Phenotypic markers were labelled using anti-CD4-FITC or anti-CD4-PerCP (both BD Bioscience) and anti-KJ1.26-APC (eBioscience) before the cells were fixed and permeabilized employing the FOXP3 Fix/Perm buffer set according to eBioscience protocols^{40 (link)}. Cells were then labelled using anti-IL-17A-PerCP or APC/Cy7, anti-IFN-γ-APC or PeCy7, anti-IL-4-PE, anti-IL-10-PE and anti-FOXP3-APC (all eBioscience) antibodies for 30 min prior to flow cytometry. Data were acquired using a FACSCanto immunocytometry system (BD Pharmingen) with samples gated according to appropriate isotype and fluorescence minus one controls and analysed using FlowJo software (Tree Star Inc, OR, USA, version 7.6.1). The gating strategy for the adoptive transfer model is shown in Supplementary Figure 2 whilst those for analysis of DLN cells from CIA mice are shown in Supplementary Figures 3 and 4.

PBMCs were isolated from peripheral blood by Ficoll gradient centrifugation (DAKEWE, Shenzhen), CD4⁺CD25^high Treg cells and CD8⁺ T cells were sorted from PBMCs by flow cytometry through signalling of appropriate antibodies: anti‐CD4‐FICT (Biolegend), anti‐CD25‐APC (Biolegend) and anti‐CD8‐Percp‐cy5.5 (Biolegend), respectively. The antibodies were incubated at 4°C for 30 minutes in the dark. Flow cytometric analyses were performed using Fluorescence Activated Cell Sorter (FACS) Canto II (BD Biosciences, Heidelberg, Germany), and the results were analysed with BD FACSDiva Software version 6.1.2. For detection of surface molecules of CD4⁺CD25^high regulatory cells, PBMCs were incubated with the appropriate mAb: anti‐CD4‐FITC (eBioscience) and anti‐CD25‐PE (eBioscience). For intracellular staining including the staining of Foxp3, HO‐1, IL‐10, LAP and CTLA‐4, cells were fixed, permeabilized and stained according to the manufacturer's instruction. The following mAb were used: anti‐Foxp3‐APC (eBioscience), anti‐HO‐1‐PE (Abcam), anti‐IL‐10‐PE (eBioscience), anti‐LAP‐PerCP (eBioscience) and anti‐CTLA‐4‐PE (Biolegend).