Anti cd11b macs beads

CD8⁺ T cells were positively selected from spleens of naive 8- to 12-week-old C57BL/6 female mice using Miltenyi MACS Enrichment according to the manufacturer’s protocol. Enriched CD8⁺ T cells were activated in vitro for 72 h using 1 µg/ml anti-CD3 and 1 µg/ml anti-CD28 to induce Fas expression. Tumor-infiltrating G-MDSCs from DIO mice were sort-purified on a BD FACSMelody after being pre-enriched using anti-CD11b MACS beads from Miltenyi, as per the manufacturer’s protocol. Prior to co-culture, purified G-MDSCs were incubated in the presence or absence of 50 µg/ml anti-FasL neutralizing antibody for 1 h. In vitro activated CD8⁺ T cells were then co-cultured with G-MDSCs in the continued presence or absence of anti-FasL for 24 h. Cells were harvested and stained for flow cytometry to evaluate CD8 T cell apoptosis as indicated above.

Cell trace-labelled CD8⁺ T cells specific for OVA (OT-I) were cultured with sorted monocytes or MO-DCs from the spleens of PbA-infected mice at different APC/T cell ratio (1:3, 1:10 and 1:30) in the presence of 40 μg ml⁻¹ of OVA protein (OVA grade V, Sigma-Aldrich). For MO and MO-DC sorting, cells from the spleens 5 days after PbA infection were pre-enriched using anti-CD11b MACS beads and LS MACS Separation Columns (Miltenyi Biotec). MO-DCs and monocytes were then sorted as CD11b^hiF4/80^hiDC-SIGN^hiMHCII⁺CD11c⁻ and CD11b^hiF4/80^hiDC-SIGN⁻MHCII⁻CD11c⁻, respectively. Splenic naive OT-I transgenic T cells were pre-enriched by negative selection using Mouse CD8⁺ T Cell Isolation Kit (Stemcell Technologies) and then sorted as CD8⁺CD62^hiCD44^low/−. Sorted naive OT-I cells were labelled with 1.25 μM of Cell Trace Violet (Invitrogen) and added to 96-well round-bottom plate at 30,000 per well. After 3 days, OVA-specific proliferation of live (Fixable Viability Dye negative, eBioscience) OT-I cells was evaluated by Cell Trace Violet dilution and staining with monoclonal antibody to CD8. Sandwich kit ELISA (Biolegend) was used to measure IFNγ in the supernatant co-cultures of OT-I and monocytes or MO-DCs according to the manufacturer's recommendations.