In vitro Polarization of CD4+ T Cells with BmEOS
Spleens were removed from C57bl/6J 6~8-week-old mice, mechanically disrupted, treated with RBC lysis buffer and run through 70 μm cell strainer. Suspensions were resuspended in MACS buffer, labelled with an antibody cocktail and naïve CD4+ T cells were negatively separated using the mouse Naïve CD4+ T cell Isolation kit (Miltenyi Biotech), in accordance with manufacturer’s instructions. High purity isolated naïve CD4+ T cells (1*10^6/ml) were co-cultured with the BmEOS (5*10^5/ml) in supplemented IMDM medium with or without 100ug/ml HDM in the presence or absence of 50 ng/ml IL-25, with IL-2 and GM-CSF treatment to sustain T cells survival. As controls, CD4+ T cells alone were also cultured. Cultures were incubated for 96 h at 37°C in a 5% CO2 humidified atmosphere. After 96 h, the cells were re-stimulated for 5 h with 1X T cell stimulation cocktail (plus protein transport inhibitors) and then were harvested and stained with fluorescent antibodies for flow cytometric analysis.