Mouse na ve cd4 t cell isolation kit

CD4 T cells were purified from the splenic cells of WT BALB/c mice by a mouse naïve CD4 T cell isolation kit (Miltenyi Biotec, Auburn, CA). The purified CD4 T cells (purity >94%, as assessed by flow cytometry) were resuspended at 1×10⁶ cells/ml in RPMI 1640 medium (Mediatech Inc., Herndon, VA) supplemented with 10% FBS (HyClone, Logan, UT), 4 mM L-glutamine, 1 mM sodium pyruvate, 55 μM β-ME, 10 mM HEPES, 100 units/ml penicillin, and 100 μg/ml streptomycin. The cells were stimulated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (1 μg/ml) (BD Biosciences, San Diego, CA) in 96 well flat bottom plates and treated with cicaprost at various concentrations or vehicle for 3 days. In T cell proliferation experiments, naïve CD4 T cells were stained with CFSE, followed by cell culture with plate-bound anti-CD3 and anti-CD28 and treatment with cicaprost (a generous gift provided by Dr. Manuela Huebner, Bayer HealthCare, Berlin, Germany) or vehicle. After 3 days of cell culture, CFSE intensity of the cells was determined by flow cytometry gated for all cells with LSR II flow cytometer (BD Biosciences). The culture supernatant was harvested for multiple cytokine ELISA assays. T cells were also harvested at day 2 for RT-PCR analyses of Bcl2, STAT1, STAT6, and β-actin mRNA expression.

To investigate the recruitment of circulating naïve T cells into TLOs, we used the mouse naïve CD4+ T cell isolation kit (Miltenyi, 130–104-453) following manufactures protocols to isolate cells from LNs and spleen of a 10–12-week-old Actin-teal fluorescent protein (TFP) mouse. A retro-orbital injection placed purified naïve CD4 T cells intravenously (1×10⁶ cells per mouse) into TNF^ΔARE/+ mice or littermate controls, both on a CD45.1 congenic background, harvesting tissue 2 hours after transfer. Intravenous injection of anti-CD45-APC-Cy7 (5μg per mouse) before endpoint allowed distinction of cells within the blood and recruited cells.
In experiments testing Treg development, 14 to 16-week-old TNF^ΔARE/+ mice and littermate controls on a CD45.1/CD45.2 congenic background were cohoused with C57/BL6 mice from a Helicobacter spp. positive colony for 4 weeks. In the 3^rd week of co-housing, mice also received 3 gavages, every other day, of H. typhlonius and H. apodemus-containing feces and were confirmed positive for the bug at the end of the week. Naïve T cells from CT2 Foxp3-GFP CD45.1 Tg mice and CT6 Foxp3-GFP CD45.2 Tg mice were sorted using an BD FACSAria™ III Cell Sorter (BD Biosciences), gated on Live / CD45⁺ / B220⁻ / TCRVa2⁺ / CD4⁺ / CD62L⁺ / CD44⁻ / Foxp3GFP⁻ cells. Sorted cells were adoptively transferred i.v. (1×10⁵ cells per mouse) 2 weeks before tissue harvesting.