Hepes buffer

Groups of age-matched C57BL/6 recipients received costimulation blockade consisting of α-CD40L (CD154) mAb (MR1, 1 mg, d0) (BioXcell) with or without CTLA4-Ig (0.5 mg, d2) (Bristol Myers Squibb Pharmaceuticals, Princeton, NJ) and 20×10⁶ unseparated BM cells recovered from naïve BALB/c donors (d0). BM cells were collected in M199 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 4μg/ml Gentamicin Sulfate (MP Biomedicals, Santa Ana, USA) and 10mM Hepes Buffer (MP Biomedicals) (BM medium). Selected groups were administered with a short course of rapamycin (0.1 mg, d-1, d0 and d2) (LC Laboratories, Woburn MA) and either received in vitro activated or IL-2 complex expanded Treg populations (3×10⁶) at the time of BMT or IL-2 complexes (d-4, -3 and -2; 5μg IL-2 / 25μg α-IL-2). Indicated groups of recipients were irradiated with 1Gy total body irradiation (TBI) prior to BMT (d-1) using a Xylon X-Ray unit [25 (link)]. Irradiated mice were additionally treated with or without IL-2 complexes (d3, d5; 1μg IL-2 / 5μg α-IL-2). All reagents were administered i.p. in a phosphate buffered solution (PBS) and BM cells were injected intravenously (i.v.) in BM medium.

Hydrogenated phosphatidylcholine (95%-hydrogenated phosphatidylcholine and 0.5%hydrogenated lyso phosphatidylcholine) was gifted from American Lecithin Company (Oxford, CT). Cit-HBr was kindly donated by Lund beck A/S (Copenhagen, Denmark). Diacetyl phosphate, NAG, and cholesterol were purchased from Fisher Scientific (Pittsburgh, PA). PEG 600 was purchased from Croda Inc. (Columbus Circle Edison, NJ). All other solvents were purchased as HPLC grade. Rat Primary Brain Microvascular Endothelial Cells (RPBMECs), Animal Endothelial cell medium supplemented with murine Endothelial Growth Factor (mEGF), fetal bovine serum (FBS), L-glutamine, Antibiotic-Antimycotic and gelatin-based coating solutions were purchased from Cell Biologics (Chicago, IL). Hank’s Buffered Salt Solution (HBSS) was purchased from Thermo Scientific (South Logan, UT). CellTox Green Cytotoxicity Assay kit was gifted by Promega Corporation (Madison, WI). D-Luciferin fluorescence dye was purchased from BD Bioscience (Bedford, MA). Trypsin was purchased from Gibco (Grand Island, NY). HEPES buffer was purchased from MP Biomedical (Solon, OH). Polystyrene nonpyrogenic 96-well plates were obtained from BD Bioscience (Bedford,MA). 24-well trans inserts obtained from Corning Life Sciences (Lowell, MA.).