Apc cy7 anti mouse cd11b

Flow cytometry analysis was performed as reported with modifications^{59 (link)}. Briefly, the upper side of the heart, containing aortic valve was isolated and cut into small pieces by using scissors and incubated for 30 min at 37 °C with stirring in RPMI1640 medium (Sigma-Aldrich) containing 2% (vol/vol) newborn calf serum (Equitech Bio) and 0.5 mg/mL collagenase (Wako Chemicals). Cell suspensions were filtered through cell strainers (70 μm; BD Bioscience), and stained with following mAbs: PE-Cy7 anti-mouse F4/80 (BioLegend; 1:100), APC-Cy7 anti-mouse CD11b (BioLegend; 1:100) and BV421 anti-mouse CD4 (BioLegend; 1:100) to detect macrophages. Anti-CD16/32 mAb (BioLegend; 1:100) and 7-AAD (BioLegend; 1:100) were used to prevent nonspecific staining and detect dead cells, respectively. Samples were analyzed by FACSAria II (BD Bioscience). Data were analyzed by using FlowJo 9.9 (Tree Star).

Mononuclear cell suspensions of spinal cords were prepared according to a standard protocol [29 (link)]. Briefly, spinal cord tissue was homogenized with 1X HBSS in the presence of Golgi Plug (1:1000, BD Biosciences, San Jose, CA) and passed through nylon mesh filter for further dissociation. Monocytes from SCs were isolated from the interphase of a gradient isotonic Percoll (30/70%, Sigma) at room temperature. Single-cell suspensions from spinal cord were incubated with Fc block (CD16/32-clone 93, BioLegend) and then stained with fluorescent-conjugated antibodies for surface markers: Alexa700 anti-mouse CD45 (BioLegend), APC-Cy7 anti-mouse CD11b (BioLegend) and phycoerythrin (PE) anti-mouse F4/80 (BD Biosciences). For intracellular staining, cells were permeabilized (Permeabilization wash buffer 10X, BioLegend) and stained with rabbit anti-mouse ATX (Cayman), followed by incubation with a FITC-conjugated anti-rabbit antibody. The appropriate isotype control (rabbit IgG) was used. Data was collected on a FACsCalibur and analyzed by FlowJo software (Tree Star, Ashland, OR).