Anti ccr4 pe

Single cell suspensions were generated from murine livers as previously described (16) .
Suspensions at a concentration of 10 7 cells/ml were incubated at room temperature (45 mins) in combinations of the following antibodies in the presence of Fc blocking antibody: anti-CD45-PE, anti-Ly6G(Gr1)-PE, anti-CD11b-PECy7, anti-CCR5-PE, anti-CXCR3-APC, anti-CXCR4-APC, anti-CXCR5-APC, anti-CD19-PE, anti-CD3-APC, anti-NK1.1-APC (all eBioscience), anti-CCR1-PE, anti-CCR2-PE, anti-CXCR2-APC, anti-CXCR6-APC, anti-CXCR7-APC (all R&D systems), anti-CCR3-PE and anti-CCR4-PE (both Biolegend). Analysis was performed using FlowJo software. The percentage of positive cells within samples was determined and the total number of cells of interest per organ was calculated.
For sorting of CD45 + /CXCR2 + neutrophils from normal and tumor-bearing livers, single cell hepatic suspensions pooled from 6 tumor-bearing or control mice were stained with anti-CD45-PE and anti-CXCR2-APC as above, before being FACS sorted using a high speed cell sorter (Beckman Coulter).
In-vivo administration of the neutrophil depleting antibody 1A8 may mask the Ly6G epitope making it impossible to subsequently identify neutrophils. We therefore used a combination of CD45 and CXCR2 to identify neutrophils in animal experiments where neutrophil depletion was performed.