Magcellect mouse cd4 t cell isolation kit

Gastric mucosa was collected by scraping using a glass slide and then strained (100 μm nylon mesh). Tissue was digested 1 h at 37°C in a PBS containing 1 mmol/L dithithreitol, 2mM EDTA, HEPES, 10% FBS, and 100 U/ml of collagenase type VIII from Clostridium histolyticum. Cells were filtered (70 μm nylon mesh) and CD4⁺ cells isolated using the MagCellect mouse CD4⁺ T cell isolation kit (R&D Systems, Minneapolis, MN). Cells were prepared with 5% FBS and 5 μg/ml Fc Block (eBioscience) at 4°C for 5-10 minutes. Labeled antibodies (Table 2) were added (1 μl/million cells) for 30 minutes at 4°C in the dark. Cells were washed twice with 5% FBS. For intracellular staining, cells were fixed and permeabilized in 1 ml of cytofix/cytoperm solution for 20-30 minutes at 4°C protected from light (BD Biosciences, San Jose, CA). Cells were then washed twice in 1X Perm Wash Buffer (BD Biosciences) and staining was performed in 100 μl Perm Wash Buffer with labeled antibodies as above. Cells were washed and suspended in 350 μl and analyzed using a LSRII flow cytometer (BD Biosciences) gated on live cells based on forward and side scatter, or using a live/dead fixable marker (LIVE/DEAD Fixable Dead Cell Stain Kit; Invitrogen). Data were analyzed using FlowJo7 software (Tree Star). Median fluorescence intensity was calculated as the difference between stimulated samples and unstimulated controls.