Ly6g percp cy5

Single cell suspensions extracted from the lungs were stained with a live/dead-Infrared marker (Thermo Fisher) and incubated with monoclonal antibody CD16/CD32 Becton Dickinson Biosciences (BD) to block Fc receptors, before adding a cocktail of directly conjugated mAbs directed against the following surface markers; CD11b-BV711 (M1/70, 563168), Ly6G-PerCp-Cy5.5 (1A8, 560602), CD45-BB515 (30-F11, 564590), SiglecF-PE-CF594 (E50-2440, 562757), CD103-BV421 (M290, 562771), CD11c-BV510 (HL3, 562949), CD3e-BV786 (145-2C11, 564379), CD19-BV605 (1D3, 563148), CD335-BV605 (29A1.4, 560469), and Ly6C-APC (AL-21, 560595) all purchased from BD and FceR1-PE-Cy7 (MAR-1, 25-5898-82), MHC-II-AF700 (M5/114.15.2, 56-5321-82) from Thermo Fisher (ebioscience). Data acquisition was performed with the BD LSR Fortessa (BD) and Diva software, and the analysis was performed with FlowJO X software (TreeStar, Ashland, OR). In initial control experiments, the right lobes were evaluated for the frequency of contaminating peripheral blood mononuclear cells after cardiac PBS infusion by staining with an anti-CD115 mAb (T38-320, 565249, BD), which is uniformly expressed on all circulating blood monocytes (13 (link)) but not on lung monocytes (14 (link)). The frequency of CD115⁺ cells was below 0.01% among the lung cells, arguing that we had little if any blood leukocyte contamination (Figure S1B).

Mice subjected to spinal cord injury were killed by an overdose of anesthetic and their spinal cords, processed for flow cytometry after saline transcardial perfusion. The lesioned cord segments extending between T11 and T13 were homogenized and reduced to single-cell suspensions by Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) according to the manufacturer’s instructions. Cell suspension was filtered through a 70-μm strainer, centrifuged over a 30% discontinuous Percoll Gradient (GE Healthcare) and myelin debris removed from the surface (modified from Cardona et al., [28 (link)]). Cells were labeled with Zombie Aqua™ die (Biolegend), then incubated with 5% FBS, 1%BSA, and 5 mg/ml rat anti-mouse Fc III/II receptor (CD16/CD32) blocking antibodies (BD), and then stained using the following monoclonal antibodies: CD45-Pb (Biolegend), CD11b-PeCy7 (BD), Ly6C-APCCy7 (BD), Ly6G-PerCP Cy5.5 (BD), and CD11c-APC (ebioscence). Cells were analyzed by LSR Fortessa (Beckton Dickinson) and data analyses by FlowJo (Treestar) software.

Peritoneal lavage cells were stained with fluorescently conjugated antibodies to MARCO-fluorescein isothiocyanate (FITC) (MCA1849F; Bio-Rad), CD11c⁻ BV605 (N418), CD86⁻ BV421 (GL-1), CD103⁻ BV510 (M290; BD Biosciences), CD11b⁻ phycoerythrin (PE)-Cy7 (M1/70), Ly6G-PerCP Cy5.5 (1A8), F4/80-APC (BM8), CD45⁻ AF700 (30-F11), MHCII-APC-Cy7 (M5/114.15.2), CD200R⁻ PE (123908), Ly6C-PE-Texas Red (AL-2; BD Biosciences), and NK1.1-BV650 (PK136) and collected on a Fortessa flow cytometer (BD Biosciences). Viability was assessed using 4′,6-diamidino-2-phenylindole (DAPI). Antibodies were purchased from BioLegend unless otherwise stated. Cells were gated based on live cells and small peritoneal macrophages (CD45⁺ F4/80⁺ CD11c⁻ CD11b⁺ MHCII⁺), large peritoneal macrophages (CD45⁺ F4/80⁺ CD11c⁻ CD11b⁺ MHCII⁻), interstitial macrophages (CD45⁺ Ly6C⁻ F4/80⁻ CD11b⁺ CD11c⁺ MHCII⁺), Ly6C-negative monocytes (CD45⁺ Ly6C⁻ F4/80⁻ CD11b⁺ CD11c⁺ MHCII⁻), CD103⁺ dendritic cells (DC) (CD45⁺ Ly6C⁻, F4/80⁻, CD11b⁻, MHCII⁺, CD103⁺ CD11c⁺), C11b⁺ DC (CD45⁺ Ly6C⁺ F4/80⁻ CD11b⁺ MHCII⁺ CD11c⁺), neutrophils (CD45⁺ Ly6C⁺ F4/80⁻ CD11b⁺ MHCII⁻ Ly6G⁺), Ly6C-positive monocytes *CD45⁺ Ly6C⁺ F4/80⁻ Ly6G⁻ CD11b⁺ MHCII⁻), plasmacytoid DC (CD45⁺ Ly6C⁺ F4/80⁻ CD11b⁻ CD11c⁺ MHCII⁺), and natural killer cells (CD45⁺ Ly6G⁻ F4/80⁻ NK1.1⁺).