Lsr 2 sorb

Bone marrow cells were isolated by crushing hind leg bones and (if required) spinal cord in cold RPMI medium, and they were treated with red blood cell lysis buffer (Sigma). Bone marrow was stained with anti-CD135-PE monoclonal antibody (mAb) or anti-CD48-PE mAb (both eBiosciences), or anti-IL-10R-PE mAb (or corresponding isotype control) (BioLegend), anti-CD34-AF700 mAb (eBiosciences), anti-CD150-PE-Cy5 mAb, anti-Sca-1-PE-Cy7 mAb, anti-c-Kit-APC-Cy7 mAb (all BioLegend), and anti-lineage-APC (BD Pharmingen). For proliferation staining, cells were permeabilized with Cytofix/Cytoperm and stained with anti-Ki-67-FITC mAb, corresponding isotype control (all BD Pharmingen), and Hoechst 33342 nucleic acid stain (from Life Technologies).
Blood was stained with anti-B220-PacBlue mAb, anti-CD11b-APC-Cy7 mAb (both BD Pharmingen), anti-Ly6/G-PE-Cy7 mAb (BioLegend), and anti-CD3-AF647 mAb (Caltaq) and treated with red blood cell lysing solution (BD Pharmingen).
Unspecific Fc-receptor binding was blocked by anti-CD16/CD32-unconjugated mAb (BD Pharmingen) prior to surface staining.
Cell counts were determined using AccuCheck counting beads (Life Technologies).
Cells were measured by flow cytometry (FACS) (LSR II Sorb; Becton Dickinson), and data were analyzed with the FlowJo software.

Mononulcear cell isolation from the liver was performed as previously described (41) . After in situ perfusion and in vitro digestion of the liver, cells were filtered through 70 µm cell strainers and centrifuged at 300 g for 8 min at 4°C. The cell pellet was resuspended in 33% Percoll and centrifuged at 500 g for 15 min at room temperature. Following red blood cell lysis (Sigma) cells were immunolabeled with fluorochrome-conjugated antibodies (Biolegend and BD) for flow-cytometry analysis (LSR II Sorb, Becton Dickinson) or cell sorting (MoFlo XDP, Beckman Coulter or FACSAria fusion sorter Becton Dickinson).