Cytocfix cytoperm kit

For cell surface molecules staining, DCs (1 x 10⁶ cells/well) were harvested, washed with FACS washing buffer (0.5% FBS and filtered 0.05% NaN₃ in PBS). The cells were then labeled for 20 min at room temperature with APC-conjugated anti-mouse CD11c (N418) and one of the following PE-conjugated mAbs: anti-mouse IA^b (AF6-120.1; MHC II), anti-mouse CD40 (3/23), anti-mouse CD80 (16-10A1) and anti-mouse CD86 (GL1) The mean fluorescence intensity (MFI) of each surface molecules was measured from the total cell population via BD accuri C6 plus flow cytometer (BD Biosciences). For intracellular staining, cells were stimulated for 5 h with 50 ng/ml of PMA, 1 μg/ml of ionomycin and Golgiplug. The cells were harvested, washed with FACS buffer and stained with FITC-conjugated anti-mouse CD4 (RM4-5) for 20 min at room temperature. The cells were fixed for 20 min using a Cytocfix/Cytoperm kit (BD Biosciences). Intracellular cytokines were detected with APC-conjugated anti-mouse IFN-γ (XMG1.2) and IL-17 (eBio17B7), PE-conjugated anti-mouse IL-4 (11B11). Treg cells were firstly stained with FITC-conjugated anti-mouse CD4 (RM4-5) and PE-conjugated anti-mouse CD25 (PC61), followed by APC-conjugated anti-mouse FoxP3 (FJK-16s) according to the manufacturer’s instructions of FoxP3 staining buffer set (eBioscience, San Jose, CA).

Mice were anesthetized and bronchoalveolar lavage fluid (BALF) was collected by 1 ml of PBS twice through the trachea. The BALF cells and lung lymphocytes were harvested, washed with FACS buffer and stained with FITC-conjugated anti-mouse CD4 (RM4-5) and CD8 (53-6.7) for 20 min at room temperature. The cells were fixed for 20 min using a Cytocfix/Cytoperm kit (BD Biosciences). Intracellular cytokines were detected with APC-conjugated anti-mouse IFN-γ (XMG1.2). The stained cells were analyzed by using FACS Accuri with Accuri C6 software (BD Biosciences).