Anti ly6g fitc

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).

Flow cytometry was performed the same day as collection to determine cell populations in whole blood, peritoneal lavage, abdominal wall, and liver tissue. Tissue was dissociated to obtain a single-cell suspension by Liberase TM research-grade solution (1:100, Roche, Basel, Switzerland) in RPMI medium and filtration through a 0.3 μm membrane filter (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s protocol. Tissue cell suspension and peritoneal lavage were centrifuged at 380 RCF for 5 min at RT to obtain a cell pellet, which was then resuspended in FACS buffer to a final concentration of 10⁶ cells/mL for further analysis. Cells in the single cell suspensions were blocked with 10% mouse serum for 10 min at 4 °C to prevent unspecific binding. Cell suspension and full blood were incubated for 15 min at 4° with the combination of fluorochrome-conjugated antibodies: anti-CD45-APC-Vio770 (1:50, clone REA737), anti-Ly-6G-FITC (1:50, clone REA526) (both Miltenyi Biotec, Bergisch Gladbach, Germany), and propidium iodide solution (1 μg/mL, Miltenyi Biotec, Bergisch Gladbach, Germany). Flow cytometry was performed using MACSQuant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany), compensation was applied and cell populations with corresponding data were analyzed with FlowJo (version 10.5.3, Becton, Dickinson and Company, Ashland, OR, USA).