Ly6g pe cf594

Peripheral blood samples were collected from submandibular vein and white blood cells were isolated after treatment with red blood cell lysis buffer. Cells were suspended in 100 µL of cell staining buffer (Biolegend) and incubated for 5 min with 0.5 µL of Trustain (Biolegend). Later, the cells were incubated with fluorescent conjugated antibodies at 4 °C for 45 min. For enumeration of inflammatory cells Ly6G-PE CF594 (BD Bioscience), CD11b-APC (Invitrogen), Ly6C-FITC (BD Biosciences) and F4/80-PE (Biolegend) antibodies were used. Dead cells were identified by using 7AAD (BD Pharmingen). Flow cytometry was carried out by using Accuri C6 (BD Biosciences) cytometer by following standard protocols as per the gating strategy shown in the Supplementary Fig. S1 for the enumeration of monocytes (Ly6G^-CD11b⁺Ly6C^hi), and M1 (F4/80⁺Ly6C^hi) or M2 (F4/80⁺Ly6C^lo)^{40 (link),42 (link)}.

The composition of tumor infiltrate was determined by flow cytometry using different combinations of the following antibodies: CD45-BV605, CD11b-BV421; Ly6G-PE-CF594; Ly6C-FITC (Clone AL-21); F4/80-PECy7; CD11c-PE; CD3e-FITC; CD4-Alexa700; CD8-PE; CD19-APC-H7; CD49b(DX5)-APC and MHCII-PercpCy5.5 from BD Bioscience, eBioscience or BioLegend (San Diego, CA, USA). Cell viability was determined by Aqua LIVE/Dead-405 nm staining (Invitrogen). Cells were analyzed on LSR Fortessa (BD Bioscience).

Peritoneal exudate cells (PECs) were isolated via lavage and single cell suspensions were briefly washed with PBS prior to red blood cell lysis using ACK lysis buffer. Prepared cells were stained with Zombie Yellow (BioLegend) to assess live status of cells. The following antibodies were used, though not necessarily all in the same panel: F4/80- BV785 (BioLegend; Clone BM8), CD115-BV421 (BioLegend; Clone AFS98), CD11c-BV421 (BD Bioscience; Clone HL3), CD115-BUV395 (BD Bioscience; Clone AFS98), CD19-BUV737 (BD Bioscience; Clone 1D3), CD226-PE (BD Bioscience; Clone TX42.1), CD11b-PE (eBioscience; Clone M1/70), MHCII (I-A/I-E)- PE/Cy7 (BioLegend; Clone M5/114.15.2), Ly6G-PE/CF594 (BD Bioscience; Clone 1A8), Ly6C-APC/eFluor780 (eBiosceince; Clone HK1.4), CD102-Alexa Fluor 647 (BioLegend; Clone 3C4 (mlC2/4)), CD102-Alexa Fluor 488 (eBioscience; Clone 3C4 [mlC2/4]), CCR2-FITC (BioLegend; Clone SA203G11), and CD11b-PErCP/Cy5.5 (BioLegend; Clone M1/70). Antibody cocktails were prepared in FACs staining buffer (phosphate-buffered saline, 1% FBS, 1% 0.5mM EDTA). Cellular fluorescence was measured using an LSRII Fortessa flow cytometer, and data were analyzed using FlowJo software (Tree Star).