Anti ccr5 pe

Blood and BM samples from killed mice were blocked for 15 min at 4 °C in PBS containing 5% BSA and a 1:100 dilution of anti-CD16/CD32 (24g2, BD Pharmingen, 553142). Samples were then stained for 30 min at 4 °C with anti-CD11b 647 (eBiosciences, 51-0112-82) or biotinylated (BD Pharmingen, 51.01712 J), anti-CD45 V450 (eBiosciences, 48-0451-82), anti-Ly6C FITC (BD Bioscience, 553104) or APC (BD Pharmingen, 560595), anti-CCR5 PE (eBioscience, 12.1951-12), and anti-CCR2 (Biolegend, 150607) primary antibodies and with streptavidin PE (BD Bioscience, 554061) secondary reagent. Erythrocytes in blood samples were lysed with FACS Lysis Solution (BD Biosciences, 349202) for 7 min at room temperature. Before gating, granulocytes were excluded by FCS/SSC. Peritoneal macrophages were collected on the indicated days after TG administration and blocked with BSA/anti-CD16/CD32. Macrophages were then stained for 30 min at 4 °C with anti-CD11b 647 (eBiosciences, 51-0112-82), anti-CD45 V450 (eBiosciences, 48-0451-82), anti-F4/80 Pe-Cy7 (Biolegend, 123114), anti-AIM (GeneTex, GTX37448), and anti-CD36 (Cascade BioScience, ABM-5525) antibodies; for quantification of dead cells, Hoechst 33258 (Sigma, 861405) was added 5 min previous to flow cytometry analysis. Data were acquired in a FACSCanto III cytometer (BD) and analyzed using FlowJo software (Tree Star).

For leukocyte surface marker determination, pooled LNCs or splenocytes were obtained from mice (as described in the mouse lymphocyte proliferation assay). Cell suspensions were prepared as described previously [31 (link)]. For immune phenotyping, the following antibodies were used: anti-CD4 FITC, anti-CD8 FITC, anti-CCR5 PE, anti-B220 FITC, anti-CD11c PE, anti-MHC class II FITC, and anti-CD11b PE (all from eBioscience, San Diego, CA, USA). Stained cells were counted with a FACS Calibur flow cytometry kit (FC500, Beckman Coulter).