Anti ly6c percp cy5

Cells from lung, spleen and lymph nodes were incubated with Fc blocker for 10 min at 4 °C, and then stained with viability dye eFluor 780 (eBioscience), anti-CD45-PerCP-Cy5.5, anti-CD11b-APC, anti-F4/80-PE, anti-Ly6C-PerCP-Cy5.5, anti-Ly6G-PE, anti-CX3CR1-FITC, anti-CD4-APC, and anti-CD8-FITC (BioLegend; 1:300 vol/vol dilution) for 30 min at 4 °C. For BM phenotyping, cells were stained for anti-CD19-APC, anti-Ter119-APC, anti-CD11b-APC, anti-Ly6C/G-APC and anti-CD3-APC as lineage markers along with anti-Ly6A/E-APC-Cy7 (Sca-1), anti-CD117-PE-Cy7 (c-kit), anti-CD48-FITC and anti-CD150-PE-Cy5 (SLAM; BioLegend; 1:300 dilution) for Lin⁻Sca-1⁺c-kit⁺ LSK populations and Lin⁻Sca-1⁺c-kit⁺ CD48⁺CD150⁻ MPPs. Data were collected using FACSCanto flow cytometer (BD Bioscience). The intracellular TNF (anti-TNF-PE; 1:600 dilution) and FoxP3 (anti-FoxP3-PE; 1:300 dilution) staining was performed using Fixation Buffer (BioLegend, 420801) and Foxp3 Staining Buffer Set (Thermo Fisher, 00-5523-00) according to the manufacturer’s instruction. Information for all flow antibodies is provided in the Reporting Summary. Flow data were analyzed using FlowJo software (Tree Star).

Flank tissue samples of dermonecrotic lesions were dissected 24 h after infection, minced and incubated for 2 h (37°C, shaking) in 2 mL of HEPES-buffered saline (Sigma) containing collagenase A (1 mg/kg, Roche Applied Sciences) and dispase II (2.4 U/mL, Roche Applied Sciences). After incubation, cells were gently dissociated using a 16G needle attached to a 10 mL sterile syringe, filtered through a 70 µm mesh, and mixed with 20 mL of washing buffer constituted by HBSS (Thermo Fisher) and 0.5% BSA (Sigma). Cells were centrifuged for 5 min at 300g, supernatant was discarded, and the pellet was resuspended in 500 µL of washing buffer. The cell suspension was incubated on ice with mouse FcR Blocking Reagent (Miltenyi Biotec) for 10 min, and then incubated for 30 min on ice with the following antibodies: anti-CD45-APC/Cy7 (1:200, Biolegend), anti-Ly6G-A488 (1:200, Biolegend), anti-Ly6C-PercP/Cy5.5 (1:200, Biolegend), anti-CD11b-BV605 (1:200, Biolegend), and Fixable Viability Dye eFluor-506 (1:1,200, Thermo Fisher). Cells were centrifuged for 5 min at 300g and the pellet resuspended in 500 µL of washing buffer/2% PFA. Flow cytometry was performed on a LSR II flow cytometer (BD Biosciences). Flow cytometry data were collected and exported using BD FACSDiva software (BD Biosciences). FACS data were analyzed and plotted using FlowJo software (FlowJo LLC).