Ly6g fitc

All animals were cared for in a specific pathogen-free room and treated following the animal care protocol approved by an animal committee. Mouse LLC1 (1 × 10⁶) cells were subcutaneously injected into 8-week-old -C57BL/6 mice. After one week of inoculation, the mice were intraperitoneally administered melatonin (30 mg/kg, three times per week) for 4 weeks. The tumor volume and mice body weights were monitored every 2 days during the experiments. Tumor volume was calculated using the following equation: length × (width)2 × 0.5. To analyze tumor-infiltrating lymphocytes (TILs), freshly isolated tumor tissue was cut into small pieces and disassociated by the gentleMACS tumor dissociation kit (Miltenyi Biotec). The suspension was further treated with a RBC lysis buffer to remove red blood cells. Approximate 1 × 10⁶ isolated cells were incubated with fluorophore-conjugated antibodies including CD45-APC, CD11b-PE, CD3-FITC, CD8-APC-Vio770, Ly6G-FITC, CD4-PE-Vio770, and F4/80-APC (Miltenyi Biotec) and analyzed by the BD FACSVia flow cytometer (BD Biosciences) [43 (link)].

Before staining, cells were incubated with FcBlock reagent (anti-CD16/CD32, Biolegend, Ozyme-France) to block the FcγRII/III receptors. The cellular content of the peritoneal exudates was characterized using a combination of fluorochrome conjugated mAbs: CD45-vioblue (REA737, Miltenyi Biotec), Ly6G-FITC (1A8, Miltenyi Biotec) F4/80-APC (CI:A3-1; AbD Serotec, BioRad, Luxembourg), Ly6C-PE (HK1.4, Biolegend), and CCR3-APC-Fire (J073E5, Biolegend). At analysis, doublets and dead cells, labeled with 7-AAD (Biolegend), were excluded. All samples were acquired using a MACS Analyzer (Miltenyi Biotec) flow cytometer and analyzed using FlowJo software (TreeStar, USA). The gating strategies are presented in Supplementary Material. Mammary gland cells were labeled with the following fluorochrome conjugated mAbs: anti-CD45-PECy7 (I3/2.3; Southern Biotech), Anti-Ly6G-FITC (1A8, Miltenyi Biotec), and anti-F4/80-APC (CI:A3-1; AbD Serotec) to identify leukocytes, neutrophils, and macrophages, respectively. At analysis, doublets, red blood cells, and lymphocytes were excluded based on CD45 staining. All samples were acquired using a CytoFLEX (Beckman Coulter, USA) flow cytometer and analyzed using CytExpert software (Beckman Coulter, USA).