Anti ly6g conjugated to pe

The kidney and bladder tissues were sliced with a scalpel into many small pieces and digested for 30 min at 37°C with 0.5 mg/ml collagenase (Sigma-Aldrich) and 100 g/ml DNAse I in PBS. After digestion, tissues were mashed and single cell suspensions were filtered through a 70 μm nylon mesh and washed with PBS. Single cell suspensions were incubated with following antibodies: anti-CD11b conjugated to APC (eBioscience, San Diego, CA, USA), anti-Ly6G conjugated to PE (eBioscience). Cells were analyzed on a FACSCanto II Flow Cytometer (BD Biosciences) using the Flow Jo software (FlowJo, Ashland, OR, USA).

The colon tissues were sliced with a scalpel into many small pieces and digested for 30 min at 37°C with 0.5 mg/ml collagenase (Sigma-Aldrich) and 100 g/ml DNAse I in PBS. After digestion, tissues were mashed and single cell suspensions were filtered through a 70 μm nylon mesh and washed with PBS. Then we separated mononuclear cells with 40% percoll. Single cell suspensions were incubated with following antibodies: anti-CD11b conjugated to APC (eBioscience, San Diego, CA, USA), anti-Ly6G conjugated to PE (eBioscience), anti-F4/80 conjugated to FITC (eBioscience, San Diego, CA, USA), anti-CD45 conjugated to APC (eBioscience, San Diego, CA, USA), anti-CD4 conjugated to PE (eBioscience), anti-CD3 conjugated to FITC (eBioscience), Cells were analyzed on a FACSCanto II Flow Cytometer (BD Biosciences) using the Flow Jo software (FlowJo, Ashland, OR, USA).