Glutamine

Enriched or sorted CD4⁺ T cell subsets were incubated in triplicate at the concentration indicated for each experiment in 96-well plates and stimulated for 2 days with 1 μg/ml plate-bound anti-CD3 mAb (145-2C11), and 1 μg/ml soluble anti-CD28 (37.51) mAb (BD Biosciences). Cells were incubated in RPMI (Invitrogen) with 5% fetal bovine serum (Intergen), HEPES (Gibco BRL), glutamine, penicillin, streptomycin (Irvine Scientific), and 2-mercaptoethanol (Sigma-Aldrich). For cytokine analysis, 100 [.proportional]l of culture supernatant was collected from each well and concentrations of IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α were determined by Flow Cytometry using the Th1/Th2/Th17 Cytometric Bead Array following the manufacturer's instructions (BD Biosciences). IL-22 and TGF-β were measured using ELISA kits according to the manufacturer's instructions (eBioscience). For intracellular cytokine staining, for the last four hours of culture, 1 [.proportional]l of BD GolgiPlug per ml medium was added to each culture and swirled gently to mix thoroughly. Cultured cells were washed twice and labeled with anti-CD4 mAb. Intracellular IL-4, IL-10, IL-17A, TNF-α, IL-2, IL-6, IFN-γ, and IL-22 content was determined by Flow Cytometry using cytokine-specific antibodies according to the manufacturer's instructions (BD Biosciences).

Sorted CD4⁺ T cell subsets were labeled with CFSE
(Molecular Probes) using the standard published protocol [29 (link)] before culture, and incubated
in duplicate at 10⁵ cells per well in 48 well plates for 48 and 72
hours with 10 μl per well anti-CD3/CD28 coated beads (BD Biosciences),
10% human serum (Gemini), HEPES (Gibco BRL), glutamine, penicillin,
streptomycin (Irvine Scientific), and 2-mercaptoethanol (Sigma-Aldrich) in RPMI
(Invitrogen). Different plates were set up for different time points. For
cytokine analysis, 100 μl of culture supernatant was collected at 48
hours and concentrations of IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IFN-γ
and TNF-α were determined by Flow Cytometry using the Cytometric Bead
Array following the manufacturer’s instructions (BD Biosciences Human
Th1/Th2/Th17 CBA kit and Biolegend LegendPlex Human Th2 panel). For cell
proliferation, cells were washed twice after 72 hours of culture and CFSE
dilution determined by Flow Cytometry. FCS Express 6 from De Novo Software
(Glendale, CA) was used to calculate proliferation index and percent divided
cells.