Pan t cells isolation kit 2

Bone marrow derived dendritic cells (BMDC) were generated from RIG-I^+/+ and RIG-I^-/- mice and T cell re-stimulation experiments were performed as previously described[31 (link)], [32 (link)]. Briefly, BMDC were first infected with PR8 at an MOI of 0.5 for 5h, washed with PBS 3 times to remove free virus, and resuspended in Iscove's Modified Dulbecco's Media complete media with FBS (IMDM, Invitrogen). For some experiments, infected BMDC were treated with IFN-I (200U/ml) before T cells were added. T cells from naïve or IAV infected RIG-I^+/+ and RIG-I^-/- mice (day 7 post infection) were enriched using Pan T cells isolation kit II (Miltenyi Biotec) and co-cultured with PR8 infected BMDC at a ratio of 10:1 for 2–3 hours followed by the addition of Brefeldin A (5μg/ml; eBiosciences). DC-T cell co-cultures were further incubated for an additional 8-10hrs. The cells were first surface stained for cell surface markers, followed by intracellular staining for cytokines. For intracellular staining, cells were incubated in permeabilization and fixation buffer (BD Pharmingen) for 45 minutes followed by 2 washes in 1xPBS, 1%FBS, 0.5% Saponin (Sigma, St Louis, MO) and intracellular staining with anti-IFNγ (2μg/ml, XMG1.2) (Biolegend), anti- Granzyme B (2μg/ml, GB11) (Biolegend), anti-TNFα (2μg/ml, MP6-XT22) (eBiosciences) for 30 minutes.

PMN-MDSCs were isolated from Balb/C mice injected with 4T1 breast tumor cells and treated with NaCl (Ctrl) +/− doxorubicin +/− GTN. At day 23, the tumors were recovered and PMN-MDSCs were isolated as previously described [17 (link)]. PMN-MDSCs were cultured alone or co-cultured for 5 days with T lymphocytes activated by anti-CD3/anti-CD28 beads and labeled with cell traceTM violet (Invitrogen, Waltham, MA, USA). T lymphocytes were previously isolated from spleens and lymph nodes of naive mice using the Pan T cells isolation kit II (Miltenyi, Bergisch Gladbach, Germany).

Isolated tumors were first dissociated and digested with collagenase I (Gibco) at 37 °C for 2 hours. Tumor homogenates were depleted from erythrocytes using ACK lysing buffer (Gibco) and filtered through 70-µm cell strainers to generate single-cell suspensions. TIL were isolated from tumor homogenates (Pan T cells isolation kit II, Miltenyi Biotec) and co-cultured with APC from spleen of naive mice in presence of antigen stimuli.