Fitc conjugated anti cd4 mab

DC from BALB/c mice were cultured with medium, F<10 kDa (20 µg/ml) or TE incomplete (without F<10 kDa, TEin, 80 µg/ml), and OVA peptide (0.5 µg/ml) in the presence or absence of LPS (1 µg/ml), and then were cultured with splenocytes from DO11.10 TCR-transgenic mice and IFN-γ production was evaluated after 5 days. Cells were stained with FITC–conjugated anti-CD4 mAb, or APC-conjugated anti-CD25 mAb (BD-Pharmingen). For intracellular IL-10, IL-4 and Foxp3 staining, the cells were cultured for 5 hours with PMA (10 ng/ml) and ionomycin (1 mg/ml; Sigma-Aldrich) and brefeldin A (10 mg/ml; Sigma-Aldrich) was added for the last 4 hours of cell culture. The cells were stained for PE- or APC-conjugated anti-Foxp3 (e-Bioscience) and PE-conjugated anti IL-10 antibody or anti IL-4 (BD-Pharmingen) using Foxp3 Fixation/Permeabilization. Concentrate and diluent and permeabilization buffer (e-Bioscience) and analyzed by FACS.

Levels of cytokines in culture supernatants were measured by ELISA. The details of the ELISA for IFN-γ and IL-4 have been described previously [28 (link), 29 (link)]. For coating and detection, the following mAbs were used: for anti-IFN-γ: R4-6A2 and XMG 1.2 mAbs; for anti-IL-4: BVD4-1D11 and BVD6-24G2 mAbs. The levels of Ag-specific cytokine production were calculated by subtracting the results of control cultures (e.g., without Ag stimulation) from those of Ag-stimulated cultures. This ELISA was capable of detecting 0.78 ng / ml of IFN-γ; 23.4 pg / ml of IL-4. For intracellular cytokine analysis, cells were incubated with ionomycin (1 μg/ml; Sigma-Aldrich) and phorbol 12-myristate 13-acetate (PMA, 25 ng/ml; Sigma-Aldrich) for 3 hr in the presence of Monensin and then stained with FITC-conjugated anti-CD4 mAb (BD Biosciences), before being stained intracellularly with PE-labeled anti- IFN-γ or IL-4 mAbs (BD Biosciences). The samples were then subjected to FACS analysis (FACS Calibur; BD Biosciences).

For cell cycle assay, cells were first exposed to IR at a proper dose. Twenty-four hours post-irradiation, the cells were then fixed in -20°C prechilled 70% alcohol overnight, and stained with 20 μg/mL propidium iodide (PI) at room temperature. For apoptosis assay, cells were harvested 24 h post-irradiation and stained with Annexin V-FITC/PI (BD Biosciences, 556547) for 30 min at room temperature. For CD4⁺CD25⁺ Treg percentage assay, cells were labeled with PE-conjugated anti-CD25 mAb and FITC-conjugated anti-CD4 mAb (BD Bioscience, 550628).
Flow cytometry assay was performed using an Amnis imaging flow cytometer (Merck Millipore, Darmstadt, Hesse, Germany) and at least 10,000 gated events were acquired from each sample. The data were analyzed with IDEAS Application v6.0 (Amnis) or FlowJo v6.0 (Tree Star, Ashland, OR, USA).