Anti foxp3 pe clone 259d c7

CD4⁺ T cells isolation from PBMCs lymphocytes were performed using human CD4 T Cell isolation kit (Miltenyi Biotec, Marburg, Germany) according to the manufacturer’s protocol. CD4⁺ T cells were stimulated with of anti-CD3/CD28 microbeads and IL-2 (20 ng/mL) and added with specific cytokine for the differentiation of specific Th cells; IFNγ (25 ng/mL) and IL-12 (25 ng/mL) for Th1; IL-6 (50 ng/mL) and TGFβ (25 ng/mL) for Th17; TGFβ (25 ng/mL) and Retinoic acid (10 nM) for Treg. Cytokine-treated CD4⁺ T cells (5 × 10⁵/well) were cultured for 5 days by adding EVs derived from naive or TSG primed WJ-MSCs in a 24 well plate. For intracellular staining, cells were stimulated with a cell stimulation cocktail (Invitrogen) for 5 h before staining. The cells were stained with cell surface marker anti-CD3-PE-Cy5, anti-CD4-FITC and anti-CD25-APC (clone M-A251, BD Biosciences) antibodies and then fixed/permeabilized and stained with fluorescence-labeled anti-IFNγ-APC (clone 4S.B3), anti-IL-17A-PE (clone SCPL1362), and anti-Foxp3-PE (clone 259D/C7, BD Biosciences) antibodies. Staining cells were analyzed by flow cytometry.

An aliquot of 100 μL of EDTA whole blood or 50 μL of tissue cell suspension was incubated in the presence fluorescent labeled anti-human cell surface monoclonal antibodies (anti-CD3-PerCP/CloneSK7, anti-CD4-PE/Clone RPA-T4, anti-CD8-FITC/CloneHIT8a, anti-CD25-APC/CloneM-A251, anti-FoxP3-PE/Clone259D/C7, all purchased from BD Bioscience, San Diego, CA, USA) to identify helper and cytotoxic T-cell subsets and Tregs. Following incubation, cells were treated with 1 mL of erythrocyte lysing solution for 10 min at room temperature. After one wash step with PBS, cells were fixed in MFF fix solution (10 g/L of paraformaldehyde, 10,2 g/L of sodium cacodilate and 6.63 g/L of sodium chloride, pH7.2). Stained cells were stored at 4 °C up to 24 h prior flow cytometric acquisition. A total of 10,000/100,000 events were acquired for each blood or tissue samples to quantify T-cell subsets and Tregs, respectively. A dual laser FACScalibur flow cytometer (488 nm and 633 nm) was used for data acquisition and storage as FCS files. The FlowJo software (TreeStar Inc., Ashland, OR, USA) was used for data analysis. The results were expressed as percentage of positive cells within the lymphocyte gate.