P-MTAB-30838 - P-MTAB-30838

Type
grow
Description
Spleens were excised and homogenised, mono-nuclear cells were isolated by density gradient centrifugation, and were seeded in 24-well plates at 4*106 cells per well in RPMI 1640 supplemented with 10% foetal bovine serum. Mouse DO.11.10 splenocytes (with CD4+ T cells expressing an ova-specific T-cell receptor) were cultured in vitro in the presence of ovalbumin (Ova), IL-2, the polarising cytokines IL-12 or IL-4, in combination with alpha-IL-4 or alpha-IFN-gamma antibodies. Briefly, spleens were homogenised, mono-nuclear cells were isolated by density gradient centrifugation, and were seeded in 24-well plates at 4×106 cells per well in RPMI 1640 supplemented with 10% foetal bovine serum. Triplicate wells were cultured in the presence of Ovalbumin (Worthington, Lakewood, NJ, USA)(10 ug/ml) and several polarising cytokine cocktails: neutral (10 ng/ml IL-2), Th1 (10 ng/ml IL-2, 10 ng/ml IL-12 and 1 ug/ml a-IL-4), or Th2 (10 ng/ml IL-2, 25 ng/ml IL-4 and 1 ug/ml alpha-IFN-gamma ). Triplicate wells were subsequently harvested on days 1, 2, 3 and 4, washed with PBS and stored in TRIzol. Naive cells (4 replicates) were harvested directly after density gradient centrifugation, washed with PBS and stored in TRIzol.
Links
Experiment E-MTAB-1475