Anti foxp3 pe cy7

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).

To assess the recruitment of Th1, Th2, and Tregs induced by treatment with ASC-derived EVs, LLN cells from mice in the asthmatic and experimental groups were cultured in anti-CD3-coated plates for 6 hours. To determine the CD4⁺CD25⁺Foxp3⁺ Treg cell populations, the cells were stained with anti-CD4-FITC (0.5 mg/ml) and anti-CD25-APC (0.2 mg/ml) according to the manufacturer's instructions (eBioscience). The cells were then fixed with a Cytofix/Cytoperm kit (eBioscience), permeabilized, and incubated with anti-Foxp3-PE-cy7 (eBioscience).
To sort the Th1 and Th2 cell populations, LLN cells were stained with the anti-CD4-FITC antibody. The CD4⁺ T cells were then stained with intracellular anti-IFN-γ-PE-cy7 (eBioscience) and anti-IL-4-PE (eBioscience) antibodies. Fluorescence was determined using a FACSCanto II cytometer (BD Biosciences) equipped with Canto software (BD Biosciences).

DCs were harvest after 24 h treatment and stained with different antibodies raised against specific surface markers: Anti-CD11C- PerCP-Cy5.5, Anti-CD86-APC, Anti-CD40-FITC, Anti-CD80-PE-Cy7, Anti-MHCII-PE, Anti-CD274 (PD-L1)-PE, Anti-CD273(PD-L2)-FITC, Anti-OX40-L-APC for 30 mins. Co-cultured cells were harvest and then mixed with different antibodies raised against specific surface markers: Anti-CD4-PerCP-Cy5.5 (eBioscience, The Netherlands), anti-CD25-FITC (eBioscience, The Netherlands), anti-CD69-PE (eBioscience, The Netherlands), anti-CCR-6-PE (eBioscience, The Netherlands), for 30 minutes. Staining for intracellular markers were performed according manufacturer’s protocol, (eBioscience, Foxp3 staining set, Bio connect, The Netherlands). Antibodies used for intracellular markers where anti-Foxp3-PE-Cy7 (eBioscience, the Netherlands), anti-RORγt-PE (eBioscience, The Netherlands). Matching Isotype controls were used to minimize the influence of nonspecific binding and proper gate setting. All incubations were performed on ice and protected from light. In total a minimum of 50,000 cells were counted and analyzed using FACS Canto II and FACS Diva software (BD Biosciences, The Netherlands).

Strains (5 × 10⁸ CFU/day/mice) were administered by intragastric gavage to WT conventional BALB/c mice for 5 days. Colon samples were removed at sacrifice and stored in RNAlater® storage solution (Ambion, Life Technologies, Foster City, CA, USA) at − 80 °C until qRT-PCR analysis. MLN and intestine were harvested and immediately processed for flow cytometry. Cell suspensions of MLN and intestine (3–5 × 10⁶ cells, in RPMI1640 supplemented by 10% FCS, 2 mM l-glutamine, 2 mM HEPES, 40 mg/ml gentamycine) were stimulated using the Leukocyte Activation Cocktail containing BD GolgiPlug (BD Biosciences) (1 μl/ml of cell suspension) for 5 h. Cells were stained by mAbs anti-mouse CD11c eFluor450, CD11b eFluor 450, B220 eFluor 450, CD3 eFluor 450, CD117 Alexa Fluor 700, NK1.1 PerCP-Cy5.5, NKp46 FITC (provided by eBioscience), CD4 APC-H7, CD90.2 BV 500; CD45RB BV 605, MHCII BV 650 (provided by BD Bioscience, san Jose, CA, USA). Subsequently, cells were permeabilized and fixed using the Transcription Factor Buffer Set (BD Bioscience), and intracellular staining was performed using mAbs anti-IL-17A eFluor450, anti-FOXP3 PE-Cy7, anti-IL-22 PE and RORgt APC (eBioscience). Flow cytometry data were analyzed using software FlowJo. Gating strategy and representative dot plots for control and BIO5768 treated mice are presented in Figs. S1 and S2.

A single‐cell suspension was stained with PE‐labelled IA^b/NP_311–325 (NIH tetramer core) at 37°C, 5% CO₂ for 2 hours in complete RMPI (RPMI with 10% foetal calf serum, 100 μg/ml penicillin‐streptomycin and 2 mM l‐glutamine) containing Fc block (24G2). Surface antibodies were added, and the cells incubated for a further 20 minutes at 4°C. Antibodies used were as follows: anti‐CD4 BUV805 (BD Biosciences; clone: RM4‐5) or CD4 APC‐Alexa Fluor 780 (eBioscience; RM4‐5), anti‐CD44 BUV395 (BD Biosciences; clone: IM7), anti‐CXCR5 BV785 (BioLegend; clone:L138D7), anti‐PD‐1 BV711 (BioLegend: 29F.1A12) and ‘dump’ antibodies: B220 (RA3‐6B2), anti‐CD8 (53‐6.7) and MHC II (M5114) all on eFluor‐450 (eBioscience). Cells were stained with a fixable viability dye eFluor 506 (eBioscience). In some cases, cells were then fixed with FoxP3 Transcription Factor Fixative kit (Thermo Fisher, UK) and stained with anti‐FoxP3 PeCy7 (eBioscience; FJK‐16S), anti‐Bcl2 FITC (Biolegend; Blc/10C4) and anti‐Ki67 BV605 (Biolegend; 16A8). Phosphorylated H3 was detected in cells fixed with 2%PFA/0.5% saponin using Alexa 647‐labelled anti‐Histone H3 (pS28) (HTA28, Thermo Fisher). Cells were acquired on a BD LSR or Fortessa and analysed using FlowJo (version 10 Treestar).