Ly6c apc cy7

Fc receptors on isolated cells were blocked with a 1:300 dilution of anti-CD16/32 (eBioscience) for 30 min at 4°C, washed with PBS-BSA 0.2%, and labeled with a 1:400 dilution of Ly-6C (APC-Cy7; BD Bioscience), CD11c (PE-Cy7; BD Bioscience), B220 (V500; BD Bioscience), CD103 (PerCP-Cy5.5; BioLegend), a 1:1600 dilution of MHCII (APC; BioLegend), and a 1:4000 dilution of CD11b (PE; BD Bioscience) for 30 min at 4°C. Cells were washed twice in PBS-BSA 0.5% and fixed in 2% PFA diluted in PBS-BSA 0.2% for 15 min at 4°C. Cells were washed, resuspended in PBS-BSA 0.2%, and analyzed by flow cytometry. Samples were collected using a BD FACS Aria III with FACSDiva software and post-acquisition analyses were performed using FlowJo.

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.

Immune cells were harvested from the airways via bronchoalveolar lavage (BAL). Briefly, mice were cannulated via a small tracheal incision and the lungs were flushed with 1 mL of sterile PBS. The collected BAL fluids were centrifuged at 1,500 rpm for 5 min at 4°C, and total cells were prepared for flow cytometry staining to determine the number and types of cells. Fc-blocked (1 μg/mL; eBiosciences) BALF cells were stained with anti-mouse SiglecF-PE (0.3 μg/mL; BD Pharmingen), CD11c-APC (0.3 μg/mL; eBiosciences), CD11b-PerCP (0.3 μg/mL; BioLegend), CD19-PECy5 (0.8 μg/mL; eBiosciences), Ly6G-PECy7 (0.8 μg/mL; BioLegend), Ly6C-APC-Cy7 (0.8 μg/mL; BD Pharmingen), and TCRβ-Pacific Blue (0.3 μg/mL; BioLegend). All samples were analyzed on a Becton-Dickinson LSR-II/Fortessa flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed by using FlowJo software (Tree Star Inc.).

Isolation of total bone marrow cells and total splenocytes was performed as previously reported (12 (link), 19 (link)). Cell pellets were then suspended in 1 ml 1 × red blood cell (RBC) lysis buffer (eBioscience, San Diego, CA) and incubated for 5 min at room temperature, followed by neutralization of the lysis buffer with 5 ml of C10 medium. This solution was further centrifuged and the pellets were resuspended in 5 ml of fresh C10. Our C10 medium is complete RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 1% 100 MEM non-essential amino acids, 10 mM HEPES, 55 μM 2-mercaptoethanol, 2 mM L-glutamine, and 100 U/ml penicillin–streptomycin (all from Life Technologies, Grand Island, NY). The resulting mononuclear cells were stained for flow cytometry as we reported previously (12 (link)). For bone marrow dendritic cell analysis, the following anti-mouse monoclonal antibodies were used: CD11c-APC, CD11b-PE, CD11b-PErCp-Cy5, Siglec-H-PerCP-Cy5.5, I-E/I-A(MHC-II)-FITC (Biolegend, San Diego, CA), and Ly6C-APC-Cy7 (BD Biosciences, San Jose, CA). For analysis of splenic T-cell subsets, we used anti-mouse CD3-APC, CD4-PE-Cy7, CD8-PerCP-Cy5.5, CD44-FITC, and CD62L-APC-Cy7 (Biolegend). Stained cells were analyzed with a BD FACSAria II flow cytometer (BD Biosciences). Flow cytometry data were analyzed with FlowJo.

Blood samples were collected through cardiac puncture or retro-orbital bleeding in heparin. Single-cell suspensions were incubated for 10 min in red blood cell (RBC) lysis buffer (Biolegend, San Diego, CA, USA) on ice to eliminate erythrocytes. Cell suspensions were incubated for 30 min on ice with anti-mouse Fc Block CD16/32 antibody (eBioscience, San Diego, CA, USA) in FACS buffer to avoid non-specific antibody binding. Cells were then washed in FACS buffer and stained with either Ig controls or fluorophore-conjugated antibodies in FACS buffer. Data acquisition was performed on a LSR II Yellow (BD Biosciences, San Jose, CA, USA) and analyzed on FlowJo version 10 (FlowJo, LLC, Ashland, OR, USA). Classical (CM: CD45⁺ CD11b⁺ F4/80⁺ Ly6G⁻ Ly6C^hi) or non-classical patrolling monocytes (PM: CD45⁺ CD11b⁺ F4/80⁺ Ly6G⁻ Ly6C⁻) were identified by FACS using the above antibodies as described [16 (link)]. Antibodies used were: anti-CD45-PerCpCy5.5 (eBioscience #45-0451-82); CD11b-eVolve 605 (eBioscience #83-0112-42); F4/80-FITC (Biolegend #123108); Ly6C-PE-Cy7 (Biolegend #128018); Ly6G-APC-Cy7 (Biolegend #127624); CD115-APC (eBioscience #17-1152-82); CD11b-PECy7 (Biolegend #101216); Ly6C-APC-Cy7 (BD-560596); Ly6G-PE (BD#551461).

We purchased directly conjugated monoclonal antibodies for flow cytometry from Invitrogen (Waltham, MA, USA): CD4-PE, Clone GK1.5; CD11b-ef450, Clone M1/70; CD44-FITC, Clone IM7; CD45AF700, Clone 30-F11; CD206-APC, Clone MR6F3; FoxP3-ef450, Clone FJK-16S, IFN-γ-PE, Clone XMG1.2; GzmB-APC, Clone GB11; iNOS-PE, Clone CXNFT; live dead fixable dead cell stain kit, Catalogue No. L34959. BD Pharmingen (San Jose, CA, USA): CD3-AF700, Clone 17A2; CD4-Pacblue and perCPcy5.5, Clone RM4-5; CD8-FITC, perCPcy5.5, and PECY7, Clone 53-6.7; CD62L-PECY7, Clone MEL-14; H2Kd-PE, Clone 17A2; Ly6G-PE and percpcy5.5, Clone 1A8; Ly6C-APCCY7, Clone- AL-21; TNF-α PECY7, Clone MP6-XT22. Biolegend (San Diego, CA, USA): CD3-PECY7 Clone 17A2; B220-percpcy5.5, Clone RA3-6B2. R&D Systems: CCR2-APC, Clone 475301; CCL2-APC, Clone 123616. eBioscience (San Diego, CA, USA): H2Kd/Dd-ef450 Clone 34-1-25; CD4-APC, Clone GK1.5.