Rat anti mouse ly6g pe

Mice were deeply anaesthetized using sodium pentobarbital (50 mg/kg) and then sacrificed by transcardial perfusion with 20 mL of cold PBS at 1 day or 3 days after PNI. The L4 DRG and spinal nerve, ipsilateral and contralateral to PNI, were removed immediately and placed in ice-cold PBS. Tissues were treated with collagenase D (1 mg/mL in RPRI 1640 containing 2% FCS, Roche, Mannheim, Germany) for 1 h at 37 °C with gently shaking. The tissues were homogenized by passing through a mesh. Cells were washed with RPRI 1640 (2% FCS) 2 times. The cells were immunostained with rat anti mouse CD11b-APC (0.4 μg/mL, clone: M1/70, Tonbo Biosciences, San Diego, CA, RRID: AB_2621556), rat anti mouse Ly6G-PE (2 μg/mL, clone: 1A8, BD Biosciences, Franklin Lakes, NJ, RRID: AB_394208), and rat anti mouse F4/80-PE (2 μg/mL, clone: BM8.1, Tonbo Biosciences, RRID: AB_2621795). Neutrophils were defined as CD11b⁺ and Ly6G⁺, while monocytes/macrophages were CD11b⁺ and F4/80⁺. The total number of neutrophils and monocytes/macrophages was analysed using a FACS Calibur (BD Biosciences) and FlowJo software (BD Biosciences).

Tumors and lungs were minced with a razor blade followed by digestion with collagenase (1 mg/mL) and DNaseI (30 μg/mL) in RPMI with 5% FBS for 1.5 hours at 37°C. Bone marrow was obtained by flushing bones with FACS buffer (2% FBS, 1% pen/strep in PBS). Single cell suspensions were subject to erythrocyte depletion with red blood cell lysis buffer (150 mM NH₄Cl, 1 mM KHCO₃, 0.1 mM EDTA) followed by two washes with FACS buffer. Cells were then stained with the indicated antibodies for 10 minutes at room temperature before flow cytometry analysis. [rat anti-mouse Gr-1-PerCP (Biolegend, #108426, clone RB6-8C5); rat anti-mouse CD11b-FITC (BD Biosciences, #553310, clone M1/70); rat anti-mouse Ly6G-PE (BD Biosciences, #551461, clone 18A); rat anti-mouse Ly6C-APC (eBioscience, # 17-5932-82, clone HK1.4)] Sorting experiments were performed on a BD FACSAriaIII cell sorter and all others were analyzed on a BD FACSCantoII. Analysis was performed using FlowJo software. Average percentages were reported with error bars representing standard error of the mean.