CD4+ T cell Isolation and In Vitro Differentiation

Mice were anesthetized with a single intraperitoneal (IP) dose of pentobarbital sodium. Following cervical dislocation the spleens were aseptically removed and a single-cell suspension was obtained by gently pressing spleens through a 40 micro molar nylon mesh cell strainer (BD Falcon, San Jose, CA) placed inside one well of a six -well cell culture plate containing 3 ml of complete media (CM): 1640 RPMI supplemented with 10% FBS, 1% glutamine (100X) in 0.85% NaCl (Invitrogen, Carlsbad, CA) 1% antibiotic-antimycotic (100X) liquid: 10,000 units penicillin (base), 10,000 µg streptomycin, 25 µg amphotericin B/ml utilizing penicillin G (sodium salt), streptomycin sulfate and amphotericin B as Fungizone® Antimycotic in 0.85% saline (Invitrogen, Carlsbad,CA). The single-cell suspension was transferred and re-filtered through the mesh nylon cell strainer into a 50-ml conical vial (BD Falcon, San Jose, CA). The single well is washed thoroughly with an additional 3 ml of supplemented complete media and also filtered through the mesh cell strainer. Isolated splenocytes were collected by centrifugation at 1200 rpm for 5 min at 40C. The red blood cells were lysed after re-suspending cells in 5 ml of ACK lysing buffer for 3 min at room temperature (RT). The buffer was neutralized with 5 ml of complete media. The resulting splenocytes were passed through a second 40 micro molar nylon mesh cell strainer and washed with complete media, pelleted and resuspended in 10 mis of complete media. The total cell number was determined, the cells were washed again and resuspended in 90 micro liter of degassed labeling buffer (solution containing PBS (phosphate buffered saline), pH 7.2, 0.5% BSA (bovine serum albumin) and 2mM EDTA (ethylenediaminetetraacetic acid)) and 10 micro liter of CD4+ (L3T4) microbeads (Miltenyi Biotec, Auburn, CA) per 107 total cells. The splenocytes were incubated on ice for 30 minutes. Subsequently, cells were washed with 10 ml of labeling buffer, spun at 1200 rpm for 5 min and resuspended in 500 micro liter of labeling buffer. The splenocytes were added to a prepared MACS LS column. After washing the column 3 times with 3 ml of labeling buffer, the column was removed from the magnetic field, 5 ml of buffer was added to the column and the CD4+ T cells were eluted from the column with the supplied plunger. The cells are counted, pelleted and resuspended in complete media at a concentration of 4 xlO6 cells per 10 ml. The purified CD4+ T cells were incubated in complete media. For THI polarizing conditions, cells were cultured with: IL-12 (2ng), IFN-gamma (100U) and anti-IL-4 (1 IBl 1, lOug) and for TH2 polarizing conditions, the cells were cultured with: IL-4 (200U), IL-6 (100U), anti-IFN-gamma (AN18, 5ug) and anti-IL-12 (clone C17.8, 2µg). All T cells were stimulated twice (day 0 and day 8) with 5 µg/ml plate-bound CD3e antibody (clone 145-2C11, BD Pharmingen), 5µg/ml soluble CD28 antibody (clone 37.51, BD Pharmingen) and IL-2 (20U) in flat-bottom 96-well cell culture plates (Corning) in tandem with polarizing conditions. Na?ve T cells were not stimulated and total RNA was extracted

Source: WO/2010/129919