Anti ly6g bv421

Blood samples were incubated with ACK lysis solution to remove erythrocytes. Single-cell suspension of splenic cells was obtained by gently pressing spleens through 70 μm cell strainers (Corning, cat. no. 352350). Bone marrow cells were harvested from one femur per mouse. Peritoneal lavage was performed by repeatedly flushing the peritoneal cavity with 10 ml PBS/2% FCS. Single-cell suspensions from the different tissues were resuspended in PBS/2% FCS and briefly incubated with Fc blocking antibodies (clone 2.4G2, purified in-house). Cells were then stained with the following fluorescently labelled monoclonal antibodies: anti-CD8α-FITC (ThermoFisher, cat. no. 11-0081-81), anti-TCRβ-PE (BioLegend, cat. no. 109208), anti-CD11b-PerCP-Cy5.5 (BioLegend, cat. no. 101228), anti-Gr-1-APC (ThermoFisher, cat. no. 17-5931-82), anti-CD19-PE-Cy7 (BioLegend, cat. no. 115520), anti-CD45-BV785 (BioLegend, cat. no. 103149), anti-CD4-BV711 (BioLegend, cat. no. 100447), anti-CD11c-BV605 (BioLegend, cat. no. 117333) and anti-Ly-6G-BV421 (BioLegend, cat. no. 127628). For nonviable cell exclusion, Fixable Viability Dye eFluor^® 780 (ThermoFisher, cat. no. 65-0865-18) was used. Stained cells were then fixed with 4% formalin and resuspended in PBS/2% FCS for flow cytometric analysis using a LSRFortessa (BD Biosciences). FACS data were then analysed with FlowJo software (RRID:SCR_008520, version 10.0.7).

Measurements were performed on a FACS Aria II (BD Biosciences, San Jose, CA, USA) and SH800 cell sorter (Sony, Tokyo, Japan), then analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Single-cell suspensions were blocked with anti-CD16/32 antibody (BD Biosciences) for 10 min, followed by staining using flow cytometry (FACS) buffer (1× PBS, 4% heat-inactivated FCS, and 2 mM EDTA) for 15 min. The antibodies for flow cytometry were as follows: anti-CD45-APC (1:50, 103112, BioLegend, San Diego, CA, USA), anti-CD45-FITC (1:50, 103108, BioLegend), anti-CD31-FITC (1:50, 102405, BioLegend), anti-F4/80-PE/Cy7 (1:50, 123113, BioLegend), anti-Ly6C-APC (1:50, 560595, BD Biosciences), anti-Ly6G-BV421 (1:50, 127627, BioLegend), anti-Sca1-APC (1:50, 108111, BioLegend), anti-CD51-PE (1:100, 104105, BioLegend), Streptavidin-PE (1:100, 405203, BioLegend), and Streptavidin-FITC (1:50, 405201, BioLegend), Annexin V-FITC (1:50, 640905, BioLegend), and 7AAD (559525, BD Pharmingen, San Diego, CA, USA).