Anti gr1 apc antibody

CD3⁺ T cells were isolated from healthy C57/B6 mice using a Miltenyi kit (Miltenyi Biotec, Auburn, CA cat. no 130-050-101). Round bottom plates were coated with 10 µg of functional CD3 antibody (eBiosciences, San Diego, CA, USA) and 5 µg CD28 antibody overnight. A total of 10⁵ CD3⁺ T cells were plated in RPMI containing 10% heat inactivated fetal calf serum, 1% penicillin/streptomycin and 1% glutamax (Invitrogen). MDSCs were sorted first by enriching the CD11b⁺ cells from the spleen and then sorted for Gr1⁺cells using a Miltenyi kit cat. no. 130-049-601. As MDSCs populations can be sorted by presence of Gr1 marker alone and are known to possess suppressive activity on day 5 [68 (link)], Gr1⁺cells were sorted using anti-APC microbeads (Miltenyi, Bergisch Gladbach, Germany) and anti-Gr1-APC antibody (BD Biosciences, San Jose, CA, USA). The purity of the sorted cell fraction was >90%. A total of 10⁵ MDSCs were incubated with CD3⁺ T cells for 72 h. Supernatants were collected at 72 h and were then subjected to IL-2 and IFN-γ ELISA assays (eBiosciences cat. nos. BMS601TEN and BMS606-2).

Bone marrow derived cells were generated as described above with the following modifications. On day 3, 100 U/ml IFN-γ (Peprotech, Rocky Hill, NJ, USA) and 0.1–1 μg/ml LPS (Sigma, St Louis, MO, USA) were added to enhance the suppressive activity of MDSCs (30 (link)). As MDSCs populations can be sorted by presence of Gr1 marker alone and are known to possess suppressive activity on day 5 (31 (link)), BMDCs Gr1⁺cells were sorted using anti-APC microbeads (Miltenyi, Bergisch Gladbach, Germany) and anti-Gr1-APC antibody (BD Biosciences, San Jose, CA, USA). The purity of the sorted cell fraction was >90%.