Luminex multiplex detection system

Mice were sacrificed one day after the final (7^th) oral challenge, and mesenteric lymph nodes were harvested. The cellular recall response was evaluated in lymphocytes isolated from mesenteric lymph nodes. Single cell lymphocyte suspensions were cultured ex vivo ± OVA (20 µg/ml) at 37°C. After 72 h, cytokine secretion was measured in cell culture supernatants using Luminex Multiplex detection system (Millipore). For real-time PCR analysis, RNA was isolated from duodenum homogenates with an RNeasy mini kit (Qiagen), and cDNA was generated with a Superscript II reverse transcription kit (Invitrogen). qPCR was performed with SYBR green master mix and commercially available primer sets (Bio-Rad). Values were normalized to GAPDH and displayed as fold induction over control samples.

Spleens and mesenteric lymph nodes (mLNs) were dissected and manually disrupted to generate single-cell suspensions. Red blood cells were depleted from splenocytes with ACK lysing buffer. Lymphocytes were resuspended in culture medium and plated at 800,000 cells per well in tissue culture-treated 96-well flat-bottom plates. Cells were cultured ex vivo with or without peanut (5 μg/mL). After 72 h, cytokine secretion was measured in cell-culture supernatants using a Luminex Multiplex detection system (Millipore, Billerica, Mass) or by ELISA (University of Michigan Cancer Center Immunology Core). For each sample, data were determined as follows:

T cell–secreted cytokines were measured from the culture supernatant after 3 d of coculture with OVA MHC II peptide–pulsed BMDCs using the Luminex multiplex detection system (Millipore, Billerica, MA).