Anti cd49b pe

Single-cell suspension from tumors were analyzed by flow cytometry as
described earlier (15 (link)). Briefly, cells
were incubated with Fc receptor blocker followed by staining with anti-F4/80 PE,
anti-CD11b APC, anti-CD206 Alexa Flour 488 (AF-488), anti-CD49b PE, anti-CD4
AF-488 and anti-CD8 PE (Biolegend, USA). Roundabout 1 (Robo1) receptor
expression was analyzed by staining with rabbit anti-mouse Robo1 antibody
(Abcam) followed by anti-rabbit AF-488 secondary antibody. For staining
intracellular targets, cells were fixed and permeablized for 30 minutes at room
temperature (Fixation/Permeablization Diluent, eBioscience). Cells were stained
with anti-EpCAM-AF-488 (Biolegend). 10–20X10⁶ cells were
recorded on FACS fortessa (BD Biosciences) and analyzed using flowJo
software.

Quadriceps muscles from immunized mice were harvested after 18–24 h of the injection and cut into small pieces. A single cell suspension was prepared using skeletal muscle dissociation kit (Miltenyi Biotec) according to manufacturer’s protocol. Briefly, small pieces of muscle were incubated in enzyme solution (enzyme D, enzyme P and enzyme A in DMEM) for 30 min at 37^oC with gentle shaking, passed through 70 µm cell strainer and wash with 10 ml DMEM. Centrifuge cell suspension at 300xg for 20 min. Cells were stained with a combination of the following fluorescently labeled antibodies: anti-CD45-AF700, anti-CD3-BV786, anti-CD19-BV605, anti-CD49b-PE, anti-CD11b-PE-Cy5, anti-CD11c-BV711, anti-F4/80-FITC, anti-Ly6C-PerCp, anti-Ly6G-PE/Cy7, anti-MHCII-PE/Dazzle594, anti-CD80-BV650, anti-CD40-APC, anti-CXCR3-BV510 and anti-CCR2-BV421 (all from BioLegend). Live/dead cells were analyzed using fixable viability dye efluor780 (eBioscience). The stained cells were acquired using BD LSRFortessa cell analyzer and data was analyzed using FlowJo software (Tree Star). Total immune cells (CD45⁺), myeloid cells (CD11b⁺), monocytes (CD11b⁺Ly6C⁺), macrophages (CD11b⁺F4/80⁺), neutrophils (CD11b⁺Ly6G⁺), dendritic cells (DCs; CD11c⁺MHCII⁺), natural killer cells (NK cells; CD49b⁺, CD3⁻), B cells (CD11b⁻CD11c⁻CD19⁺), T cells (CD11b⁻CD11c⁻CD3⁺) and NKT cells (CD49b⁺CD3⁺) were gated by flow cytometry.