Ly6c bv785

Dissociation of fresh tumor samples and antibody staining was performed as described previously^{40 (link)}. Cells were blocked with anti-mouse CD16/CD32 FC block (Biolegend, 1:100) for 10 min on ice and stained with Zombie Aqua Fixable Viability Kit (Biolegend, 1:500) to discriminate live and dead cells. The following antibody cocktails were used: CD4 BUV805 (BD, 1:100), CD3εBUV395 (BD, 1:20), CD8a BV785 (Biolegend, 1:100), CD25 BV650 (Biolegend, 1:50), TCRγ/δ BV421 (Biolegend, 1:100), CD62L PE (Biolegend, 1:500), CD44 APC-Fire (Biolegend, 1:30), CD45 PerCP Cy5.5 (Biolegend, 1:100), CD19 FITC (Biolegend, 1:100), EpCAM APC/AF647 (Biolegend, 1:200) for acquisition of adaptive immune cells; CD11c BUV737 (BD, 1:30), NK1.1 BUV395 (BD, 1:25), Ly6C BV785 (Biolegend, 1:200), CD11b BV650 (Biolegend, 1:100), F4/80 BV421/PB (Biolegend, 1:30), CD45 PerCP Cy5.5 (Biolegend, 1:100), Ly6G PE (Biolegend, 1:200), CD68 APC-CY7 (Biolegend, 1:20), EpCAM APC/AF647 (Biolegend, 1:200) for acquisition of innate immune cells. 1 × 10⁶  events were acquired per antibody panel on the BD LSRFortessa. Flow cytometry data were analyzed using FlowJo software (v10.6.2).

To analyze the skin-infiltrating cell populations, mice were infected as described above. After 3 and 6 h post infection the infected leg was shaved and amputated above the patella. As control, non-infected animals were taken. The skin was removed, minced and transferred to 10 mL DMEM buffer, containing 1 mg/mL Collagenase P (Sigma Aldrich, St. Louis, USA) and 1 U/mL DNAse I (Merck, Darmstadt, Germany). The tissue was shaken for 1 h at 37°C. After 30 min, the solution was pipetted up and down to increase digestion. Following incubation, the suspension was filtered through a 40 µm cell strainer and washed with DMEM containing 10% FCS. Cells were counted and used immediately for FACS staining. The cells were stained with Zombie Yellow (BioLegend, SanDiego, USA) for 20 min at RT, followed by surface antibody staining (CD45 AF700; 1:800 [clone DX5, BioLegend, SanDiego, USA], SiglecF PE 1:100 [clone 1RNM44N, Thermo Fisher Scientific GmbH, Germany], Ly6G APC-Cy7; 1:150 [clone 1A8, BioLegend, SanDiego, USA], Ly6C BV785; 1:150 [clone HK1.4, BioLegend, SanDiego, USA]) in Fc-Blocking buffer for 30 min at 4°C.