Anti cd25 apc

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).

Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). Human recombinant IL-6 and IL-17A, and human neutralizing antibodies to IL-6 (aIL-6) and IL-17A (aIL-17A) were obtained from R&D Systems (Minneapolis, MN, USA). The monoclonal mouse anti-human IL-6 antibody (ab9324) was purchased from Abcam (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for IL-17A, IL-6, IL-10, IL-1β, PGE₂, and TGF-β; fluorescence-activated cell sorting (FACS) human antibodies including anti-CD4-FITC, anti-IL-17A-APC, anti-Foxp3-PE, anti-CD25-APC, and their corresponding anti-mouse IgG1 K-PE/APC; and the Annexin V apoptosis detection kit were all purchased from eBioscience (San Diego, CA, USA). Antibodies against JAK2, p-JAK2, STAT3, p-STAT3, p-AKT, AKT, cyclin D2, P27, and GAPDH were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) reagents were obtained from Takara (Beijing, China).Rituximab was purchased from Novartis (Basel, Switzerland). Doxorubicin and Ara-C were obtained from Pfizer (Shanghai, China).

The Th1 and Treg cells were determined by CD4⁺IFNγ⁺, CD4⁺CD25⁺Foxp3⁺. The following antibodies were used to stain mice splenocytes: anti-CD4-FITC (eBioscience), anti-IFN-γ-APC (eBioscience), anti-CD25-APC (eBioscience), and anti-Foxp3-PE (eBioscience). Transcription factors Foxp3 were stained with antibodies after being formulated by the Foxp3/transcription factor staining buffer set. For interferon- γ (IFN-γ) detection, cells were cultured with a 1 × cell stimulation cocktail (plus protein transport inhibitors) for 4–6 h, fixed and permeabilized with a fixation and permeabilization buffer before staining with antibodies. Flow cytometry was performed on a BD CantoII cell analyzer and analyzed by FlowJo software (TreeStar). The percentage of Th1 and Treg cells was defined as Th1 and Treg cells within the CD4⁺ T cells.

To detect Foxp3, CD4⁺ T cells were incubated with anti-CD4-FITC and anti-CD25-APC (eBioscience, USA). After permeabilization and fixation, the cells were incubated with anti-Foxp3-PE or an IgG1 control (eBioscience, USA) at room temperature for 30 min in the dark. Then, the cells were then analyzed with the FACScan system.

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).

Antibodies purchased from eBioscience consisted of anti-CD4-FITC, anti-CD25-APC and anti-Foxp3-PE antibodies. The spleens were harvested, lyzed, and single-cell suspensions were prepared. Next, the cells were labeled with anti-mouse CD4 antibodies before permeabilization with Cytoperm/Cytofix (Becton Dickinson) according to the manufacturer’s instructions. After permeabilization, the cells were incubated with labeled antibodies that were specific for either mouse IL-17 or FoxP3. Then, the cells were centrifuged and the pellets were washed to remove unbound antibodies. After surface and intracellular labeling, mononuclear cells were analyzed by FACS Calibur (Becton Dickinson, USA) according to the manufacturer's instructions.