Anti ly6c bv605

Prior to antibody staining, cells were incubated for 10 min on ice with TruStain fcX (anti-mouse CD16/32, BioLegend #101319, clone 93, 1:100). To identify infiltrating monocytes/macrophages from the brain, anti-CD11b-PE-Cy7 (BioLegend, M1/70, 1:100), anti-CD45-pacificBlue (BioLegend, 30-F11, 1:100), anti-F4/80-APC (BioLegend, BM8, 1:75) and anti-Ly6C-BV605 (BioLegend, HK1.4, 1:500) were used. Blood-derived monocytes were identified using anti-CD45-pacificBlue (BioLegend, 30-F11), anti-Ly6C-BV605 (BioLegend, HK1.4, 1:100), anti-Cd115-APC (BioLegend AFS98, 1:100) and anti-Cd11c-APC-Cy7 (BioLegend, N418, 1:100). Cells were stained by incubation with antibodies for 30 min on ice. Finally, cells were washed using 1 ml PBS and centrifuged at 400 × g for 8 min. Cell pellets were resuspended in 300 µl PBS supplemented with 0.2% FBS and filtered through a 35 µm cell strainer (BD Falcon). Cell subpopulations were finally sorted into RLT buffer (Qiagen) using a BD FACSAria II SORP Cell Sorter.

Neutrophil depletion was achieved by intraperitoneal doses of 1A8 monoclonal anti-Ly6G antibody (Bio X Cell, Lebanon, NH) at 25 mg/kg in saline. Equivalent doses of IgG monoclonal anti-trinitrophenol (clone 2A3, Bio X Cell, Lebanon, NH) were administered to control mice 24 hours before carrying out the dorsal skin edema inflammation model. Four hours after dorsal skin model, mice were killed by anaesthetization, and whole blood was collected through cardiac puncture for flow cytometric analysis of neutrophils. The heparinized blood (1:100, stock heparin 5,000 IU/ml) was incubated with the following antibodies: anti-Ly6G-BV785 (1:200, catalog 127645, BioLegend, San Diego, CA) and anti-Ly6C BV605 (1:200, catalog 128035, BioLegend, San Diego, CA). Samples were acquired in an LSRFORTESSA flow cytometer (BD Biosciences, Oxford, United Kingdom) and analyzed using FlowJo software 9.7.5 (FlowJo, Ashland, OR).

For surface staining, cells were incubated with 50 µl purified anti-mouse CD16/32/FcBlock for 10 min at 4 °C, as described previously^{45 (link)}. Additional antibodies were then added in FACS buffer to a final volume of 100 µl at 4 °C for 20 min. After staining, cells were washed twice and resuspended in FACS buffer for analysis. Dead cells were excluded from analysis by fixable viability dye eFluor™ 780 (Thermo Fisher Scientific, MA). Flow cytometry was performed on an LSR Fortessa (BD Biosciences, NJ) with subsequent data analysis using FlowJo software (Tree Star). Cells were quantified by using CountBright Absolute Counting Beads (Thermo Fisher Scientific, MA). The following antibodies were purchased from Biolegend: anti-CD45.2-R-phycoerythrin-cyanine 7 (PECy7) (clone: 104), anti-MHCII I-A/I-E-AF700 (clone: M5/114.15.2), anti-CD11c-BV786 (clone: N418), anti–CD3e-PECy5 (clone: 145-2C11), anti-CD19-BV650 (clone: 6D5), anti-Ly6G-Peridinin-chlorophyll (PerCP)-Cy5.5 (clone: 1A8), anti-F4/80-AF647 (clone: BM8), anti-CD24-BUV395 (clone: M1/69), anti-CD64-PE (clone: X54-5/7.1), anti-Ly6C-BV605 (clone: HK1.5). The following antibodies were purchased from BD Biosciences: anti-CD11b-Brilliant UltraViolet (BUV) 737 (clone: M1/70).