Immunocult xf t cell expansion medium

Purified CD4⁺ T cells were co-cultured with hUCB-MSCs or exosome from hUCB-MSCs. Cells were plated at a 1:10 ratio (CD4⁺ T cells: hUCB-MSCs or CD4⁺ T cells: Exosome from hUCB-MSCs) in 24-well culture plates. Th1 cells were polarized in ImmunoCult -XF T Cell Expansion Medium supplemented with ImmunoCult Human CD3/CD28/CD2 T Cell Activator, 10 ng/mL recombinant human interleukin-12 (IL-12; Peprotech), and 5 μg/mL anti-IL-4 neutralizing antibodies (BD Bioscience) for 5 days. Th2 cells were polarized in ImmunoCult-XF T Cell Expansion Medium supplemented with ImmunoCult Human CD3/CD28/CD2 T Cell Activator, 20 ng/mL recombinant human interleukin-4 (IL-4; Peprotech), and 5 μg/mL anti-IFNγ neutralizing antibodies (BioxCell, Lebanon, NH, USA) for 5 days. Th1 and Th2 were treated with 20 ng/mL IL-2 on day 3 of polarization. Treg cells were polarized in RPMI 1640 supplemented with 10% FBS, 2 mM glutamax (Gibco), 50 μM β-mercaptoethanol, 2 ng/mL transforming growth factor beta 1 (TGF-β1; Peprotech), and 10 ng/mL IL-2 for 5 days.

Murine T cells were enriched from spleens using the EasySep CD8 T cell enrichment kit (StemCell Technologies) and cultured in RPMI-1640 media supplemented with 10% FBS, 1% penicillin–streptomycin, 1% GlutaMAX, 10 mM HEPES, 1 mM sodium pyruvate, and 55 μM 2-mercaptoethanol. B16-F10 (ATCC) and MC38 (obtained from Dr. Wucherpfennig’s lab) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The media for ovalbumin-expressing B16 cells (obtained from Dr. Wucherpfennig’s lab) was further supplemented with 0.5 mg mL⁻¹ geneticin. All cell lines were tested for mycoplasma and confirmed to be negative. All supplements were obtained from Life Technologies. Blood collars for human T cells were obtained from the Brigham and Women’s Hospital Blood Donor Center. Appropriate consent was obtained from all donors. T cells were enriched using the Rosette Sep Human T cell enrichment kit, and cells were separated via ficoll gradient separation using SepMate. T cells were cultured in ImmunoCult-XF T Cell Expansion Medium supplemented with 10 ng mL⁻¹ IL-2 (Peprotech) and activated with 25 µl mL⁻¹ ImmunoCult Human T Cell Activator (all from StemCell Technologies). The purity of the isolated cells was determined using anti-human CD3 antibody (BioLegend) and confirmed to be greater than 95% purity.