Cd8a t cell isolation kit

To generate DC, bone marrow isolated from C57BL/6 mice were cultured in 6-well plates for 7 days in presence of GM-CSF. On day 6, DC were activated overnight by 1 μg/ml lipopolysaccharide (Sigma-Aldrich). Activated DC were pulsed at 10 × 10⁶ cells/ml with 10 μg/ml of hgp100 (KVPRNQDWL) or gp33 (KAVYNFATM) peptide for 2 h at room temperature (RT). DC (5 × 10⁵) were injected subcutaneously on each flank of C57BL/6 mice. In parallel, mice were injected retro-orbitally with 2.5 × 10⁵ pMel CD8 T cells isolated from spleens and magnetically purified using CD8a + T cell Isolation Kit (Miltenyi Biotech) according to the manufacturer’s protocol. Six days later half of the mice were intraperitoneally injected with 500 μg of anti-NKG2D antibody, clone HMG2D or hamster IgG (BioXCell) [22 (link)–26 (link)], as described in the figure legend.

CD8⁺ T-cells were purified (>95%) from a cell suspension harvested from crushed spleens of OT-I mice, using a CD8a⁺ T Cell Isolation Kit and magnetic-associated cell sorting (MACS), according to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Similarly, DCs were purified (>85%) from spleens of C57BL/6 mice, using MACS CD11c microbeads (Miltenyi Biotec). T-cells and DCs were cultured immediately following isolation in a ratio of 3:1, respectively, with 1 µg/ml of ovalbumin peptide (OVA257-264, InvivoGen, San Diego, CA, USA) in a RPMI 1640 medium w/o phenol red, supplemented with 10% serum, 100 U/ml of penicillin, 100 mg/ml of streptomycin, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 50 mM β-mercaptoethanol (Biological Industries, Beit Haemek, Israel).

CD4⁺ or CD8⁺ T cells were isolated from the spleen and peripheral lymph nodes of OTII (with or without DOCK2 expression) and OTI TCR transgenic mice by magnetic sorting with Dynabeads mouse CD4 (Invitrogen) followed by treatment with DETACHaBEAD™ mouse CD4 (Invitrogen), or by a CD8a⁺ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. For antigen stimulation, OTII CD4⁺ T cells or OTI CD8⁺ T cells (1 × 10⁶ cells per well) were cultured for 2–3 days in a 24-well plate with T cell depleted, irradiated C57BL/6 spleen cells (3–5 × 10⁶ cells per well) in a total volume of 2 ml in the presence of OVA323–339 (0.5 µg ml⁻¹) or OVA257–264 (0.5 µg ml⁻¹), respectively. Viable cells were recovered by density gradient centrifugation using the Lympholyte-M cell separation media (Cedarlane, Hornby, Ontario, Canada). The purity of live OTII CD4⁺ T cells (CD4⁺Vα2⁺Vβ5⁺) or OTI CD8⁺ T cells (CD8a⁺Vα2⁺Vβ5⁺) was above 98%, as assessed by flow cytometry.

Macrophage polarization was performed as previously described (Smith et al., 2015 (link)). Briefly, 10 ng/ml PMA was first used to activate THP1 cells for 24 h, making the cells adhere to the plate wall as M0 macrophages. The M0 macrophages were further activated with LPS or IL-4+IL-13 to polarize to M1 or M2 for subsequent experiments. The polarized macrophages were washed three times with serum-free culture medium and then co-cultured with separated CD8T cells. In some co-culture experiments, an anti-hIL18 antibody (InvivoGen) was added to neutralize IL-18 in the culture. A CD8a+ T Cell Isolation Kit (Miltenyi, 130-104-075) was used for CD8T cell separation according to the manufacturer’s instructions. For spleen or tumor CD8T cell isolation, tissue homogenization was performed first. Red Blood Cell Lysis Buffer (Beyotime Biotechnology, C3702) was then used to lyse the red blood cells, followed by CD8T cell purification. Purified CD8T cells were identified by flow cytometry as >90% isolated cells with CD8⁺ phenotypes.