Pe conjugated anti h2kb

Lamina propria, lung and liver lymphocytes were isolated as described previously (31 (link)). Briefly, lamina propria lymphocytes from intestine were isolated using the Lamina Propria Dissociation Kit (Miltenyi Biotec) according to the manufacturer’s instructions followed by Percoll gradient centrifugation. Lungs were digested with collagenase D and lymphocytes were isolated by Percoll gradient centrifugation. Liver lymphocytes were isolated by Percoll gradient centrifugation. Single cell suspensions were treated with Gey’s solution and resuspended in PBS added with 2% BSA. Antibodies used for flow cytometric analysis were as follows. Percp-Cy5.5-conjugated anti-CD4 and anti-CD62L, APC-Cy7-conjugated anti-CD8 and anti-CD44, PE-conjugated anti-H2Kb, anti-CD62L, RORγt and anti-Foxp3, PE-Cy7- conjugated anti-IFNγ, anti-CD69, anti-CD8, anti-CD25 and anti-CD44, Alexa-647- conjugated anti-TNFα, FITC-conjugated anti-CD44, anti-CD4 and anti-H2kb, APC-conjugated anti-phos-Erk, anti-H2Kd and anti-IL-17 antibodies were purchased from BD Biosciences or eBioscience. In some experiments, cells were stained with an Aqua dead cell exclusion dye. Foxp3 staining kit was from eBioscience. Samples were applied to LSRII flow cytometer (Becton Dickinson), and data were collected and analyzed using FACSDiva software (Becton Dickinson).