Pe conjugated rat igg2a κ

Cells were washed twice in cold phosphate buffered saline (PBS) and resuspended at 1 × 10⁶ cells/mL in FACS buffer (PBS/0.1% NaN₃/1% FBS). Cells were blocked with rat anti-mouse CD16/CD32 (BD Pharmingen) at 4°C for 5 min and then stained for 30 min with FITC-conjugated anti-mouse-CD40 and PE-conjugated anti-mouse CD86 (BD Pharmingen) on ice in the dark. For isotype controls, FITC-conjugated rat IgG2a κ or PE-conjugated rat IgG2a κ (BD Pharmingen) was used. The cells were washed twice and resuspended in FACS buffer. Ten thousand cells were collected for each sample and analyzed on a Navios Flow Cytometer (Beckman Coulter, Brea, CA, USA). The data were analyzed with Kaluza software.

Cells were pre-incubated with PMA (5 ng/ml) and ionomycin (500 ng/ml) for 6 h, and with brefeldin A for 5 h (10 μg/ml; all from Sigma-Aldrich). For cytokine detection, FITC-conjugated anti–IFN-γ (Clone: B27, Cat# 554700, RRID : AB_395517), PE-conjugated anti-IL-5 (Clone: JES1-39D10, Cat# 559332, RRID : AB_397229), or IL-17 (Clone: SCPL1362, Cat# 560436, RRID : AB_1645514), BV711 conjugated anti-IL-13 (Clone: JES10-5A2, Cat# 564288, RRID : AB_2738731), and allophycocyanin-conjugated anti-IL-4 (Clone: 8D4-8, Cat# 560671, RRID : AB_1727546) or IL-21 (Clone: 3A3-N2.1, Cat# 562043, RRID : AB_10896655, all from BD Biosciences) were used alone or together. Isotype control Abs included FITC-conjugated mouse IgG1 κ (Clone: MOPC-21, Cat# 551954, RRID : AB_394297), PE-conjugated rat IgG2a κ (Clone: R35-95, Cat# 559317, RRID : AB_10050484), BV711-conjugated rat IgG1 κ (Clone: R3-34, Cat# 563283, RRID : AB_2869482), and Alexa Fluor (AF)-647-conjugated mouse IgG1 κ (Clone: MOPC-21, Cat# 557732, RRID : AB_396840, all from BD Biosciences).