Anti mouse ly6g ly6c

Bone marrow-derived mature megakaryocytes were generated as described [25 (link)]. Briefly, mouse femurs were flushed, and cells expressing Ly6G/Ly6C, CD11b, CD16/32, and B220 were depleted using magnetic beads (sheep anti-rat IgG Dynabeads, Thermo Fisher Scientific, Waltham, MA, USA) and the following antibodies: anti-mouse Ly6G/Ly6C (561103, BD Biosciences, Franklin Lakes, NJ, USA), anti-mouse CD11b (47-0112-82, Thermo Fisher Scientific), anti-mouse CD16/CD32 (553141, BD Biosciences), and anti-mouse B220 (BD Biosciences). Negatively selected cells were incubated in megakaryocyte medium (Stempro-34 SFM, Thermo Fisher Scientific) containing 2.6% nutrient supplement, 1% glutamine, 1% penicillin–streptomycin–fungizone, and 20 ng/mL stem cell factor (Peprotech EC Ltd., London, UK) for 2 d at 37 °C and 5% CO₂, followed by a 5-d incubation with additional 50 ng/mL thrombopoietin (TPO) (Peprotech EC Ltd.). Mature megakaryocytes were enriched with a gradient of 3%/1.5% BSA (PAA Laboratories, Fisher Scientific, Hampton, NH, USA) under gravity for 45 min at RT. Cells in the lower 25% of the gradient, representing mature megakaryocytes, were washed in PBS and harvested in TriFast™ (VWR, Radnor, PA, USA).

For murine samples, mice with tumors were killed according to the institutional ethical guidelines and femurs, tibias, and tumors were collected. Signal cell of suspension of tumor was obtained by using the Tumor Dissociation Kit (Miltenyi Biotech, Bergish Gladbach, Germany, 130-096-730) according to the manufacturer’s instructions. Antibodies (BD Pharmingen, San Diego, CA, USA) used are listed: anti-mouse-CD3e (BD Pharmingen, 566494), anti-mouse-CD45 (BD Pharmingen, 553079), anti-mouse-CD8e (BD Pharmingen, 553035), anti-mouse-CD11b (BD Pharmingen, 550993), anti-mouse-Ly-6G/Ly-6C (BD Pharmingen, 553129), Anti-mouse-F4/80 (BD Pharmingen, 565410). For cell apoptosis assays, apoptosis cells were measured by a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Data were acquired using FACS Calibur (BD Biosciences), and analyzed using Cell Quest Pro software (BD Biosciences)^{46 (link)}.