Mojosort mouse cd8 na ve t cell isolation kit

CD8⁺CD44^– T cells were purified from spleen and peripheral lymph nodes using the MojoSort™ Mouse CD8 Naïve T-Cell Isolation Kit (BioLegend, #480044) followed by CD25-depletion using the mouse CD25 MicroBead Kit (Miltenyl Biotec, #130-091-072), all according to manufacturers’ protocols. Naïve CD8⁺ T cells (1 × 10⁶/mL) were activated with plate-bound anti-CD3 (clone 145-2C11, 0.25 μg/ml, BioLegend) and anti-CD28 (clone 37.51, 0.5 μg/ml, BioLegend) in T-cell medium (high glucose DMEM, 10% FBS, penicillin/streptomycin, non-essential amino acids, HEPES, L-glutamate and sodium pyruvate) at 37°C in 10% CO₂ for 2 days. Cells were then removed from CD3/CD28 activation and re-cultured (5 × 10⁵/ml) in differentiation medium (T-cell medium supplemented with 10 IU/ml rhIL-2 from NCI) at 37°C in 10% CO₂. Cells were then sub-cultured every day in differentiation medium at 5 × 10⁵/ml each sub-culture. On day 5, cells were harvested and used for Western blots, ChIP-qPCR, qPCR or adoptive T-cell transfer.

CD8⁺ naïve T cells were isolated from spleen using Mojosort Mouse CD8 Naïve T cell isolation Kit (Biolegend) and adjusted to a concentration of 1.0x10⁶ cells/mL. Purified cells were activated with plate-bound anti-CD3 (1.5μg/mL) and complete RPMI media containing anti-CD28 (1.5μg/mL) and β-mercaptoethanol (50nM) for Tc0 polarization, or further supplemented with Tc1 [IL-2 (10ng/mL)] or Tc17 [TGFβ (2ng/mL), IL-6 (20ng/mL), anti-IFNγ (10μg/mL), IL-23 (20ng/mL), and IL-1β (5ng/mL)] polarization conditions for 72 hours (Flores-Santibáñez et al., 2018 ).

CD8⁺ naive T cells were isolated from spleen using Mojosort Mouse CD8 Naïve T cell isolation Kit (Biolegend) and adjusted to a concentration of 1.0 × 10⁶ cells/ml. Purified cells were activated with plate-bound anti-CD3 (1.5 µg/mL) and complete RPMI media containing anti-CD28 (1.5 µg/ml) and β-mercaptoethanol (50 nM) for Tc0 polarization, or further supplemented with Tc1 [IL-2 (10 ng/ml)] or Tc17 [TGFβ (2 ng/ml), IL-6 (20 ng/ml), anti-IFNγ (10 µg/ml), IL-23 (20 ng/ml), and IL-1β (5 ng/ml)] polarization conditions for 72 hr (Flores-Santibáñez et al., 2018 (link)).