Anti mouse cd16 cd32

Melanoma tissues were digested with 1 U/ml collagenase (Sigma Aldrich, Italy), passed through 70-μm cell strainers and red blood cells (RBC) were lysed to prepare single cell suspensions. Cell samples were blocked with anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA). Antibodies against CD11c-FITC, CD11b-PeCy5.5, Gr1-PE or Gr1-allophycocyanin, CD3-PeCy5.5; CD8-allophycocyanin or CD8-PE; CD4-allophycocyanin; NK1.1-PE were obtained from eBioscience and BioLegend. Expression of FAP in melanoma tissue was also analyzed by flow cytometry by using an anti-FAP-FITC and expressed as percentage of positive cells. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences).

The stromal vascular fraction from epididymal fat depots was isolated as described above. After lysing erythrocytes, cells were resuspended in FACS buffer (2% FCS in PBS) and counted on a CASY Counter. Here, 1,000,000 cells were used for cell surface staining, whereas 3,000,000 ones for intracellular staining. Co-cultures were separated using Cell Stripper (ThermoFisher) and resuspended in FACS buffer. Cell surface antigens were blocked with Anti-Mouse CD16/CD32 (1/300,14-0161, eBioscience) and stained for TCRb (1/400, PerCpCy5.5, 45-5961 Invitrogen), TCRgd (1/400, APC, 17-5711 Invitrogen), CD4 (1/400, PeCy7, 25-0041, Invitrogen), CD8 (1/400, APC-eFluor 780, 47-0081 Invitrogen), CD11c (1/400, PerCpCy5.5 45-0114 Invitrogen), CD19 (1/400, PeCy7 25-0193 Invitrogen), CD11b (1/400, PerCpCy5.5 45-0112 Invitrogen), CD11b (1/400, APC 17-0112 Invitrogen), CD11b (1/400, AF488, 53-0112-82, Invitrogen) F4/80 (1/400, PeCy7 25-4801), F4/80 (1/400, AF700, 56-4801-82 Invitrogen), and CD206 (1/400, FITC 123005 Biolegend). Staining of Tregs was done using Mouse Regulatory T Cell Staining Kit (88-8111 eBioscience). FACS measurement was done on a Canto II or LSR II (BD Biosciences) and FlowJo was used for analysis.

Tissue samples obtained at the day of lobectomy were frozen and stored at −80 °C. Frozen specimens were sectioned at 4–6 μm with a cryostat, placed on slides, air dried and fixed for 5 min with 100% acetone. Before incubation with antibodies, Fc receptors were blocked with anti-mouse CD16/CD32 (eBioscience) 10% in PBS for 30 min. Two stainings were performed: a triple-staining CD8, 103 and E-cadherin and a double-staining CD8 and CD49a using conjugated antibodies (PE labelled anti-human CD103 (eBioscience); FITC-labelled anti-human CD8 (eBioscience); AF647-labelled anti-human E-Cadherin (Biolegend) and PE-labelled anti-human CD49a (Biolegend)). Isotype-matched antibodies were used as negative controls. Nuclei were highlighted using DAPI mounting medium. The procedure and the source and concentrations of antibodies are detailed in Supplementary Table 2.
Slides of stained lung sections were scanned with an automatized fluorescent microscope Axio Scan.Z1 (Zeiss) at the magnification × 200. A coupled Zen software was used to analyse the virtual slides. For each slide, counts were done by a senior pathologist on at least five representative fields with a magnification of × 200.

Single cell suspensions were prepared from harvested melanoma tissues. Tissues were dissected and digested with collagenase 1U/ml, passed through 70-μm cell strainers and red blood cells (RBC) were lysed. Cell samples were pre-incubated with anti-mouse CD16/CD32 (eBioscience, San Diego, CA, USA) to block non-specific Fc-mediated interactions. Antibodies against CD11c-FITC, CD11b-PeCy5.5, Gr1-PE or Gr1-allophycocyanin, CD3-PeCy5.5; CD8-allophycocyanin or CD8-PE; CD4-allophycocyanin; NK1.1-PE were obtained from eBioscience and BioLegend. Data were acquired with a FACSCalibur flow cytometer (BD Biosciences).