Anti cd11b pe cy5

Bladders were digested at 37 °C for 30 min in RPMI-1640 with 10 mM HEPES, collagenase D, and DNAse (all from Sigma-Aldrich), forced through a 70 μm cell strainer (Corning), and washed with 5% fetal bovine serum (FBS, ThermoFisher Scientific, Durham, NC, USA) in PBS. Urines were diluted in fluorescence-activated single-cell sorting (FACS) Buffer (PBS supplemented with 5 mM EDTA and 3% FBS) for 30 min (pH neutralization), prior to staining. Single-cell suspensions resuspended on ice-cold FACS Buffer were stained with anti-CD45 eFluor450 (eBioscience, San Diego, CA, USA), anti-CD3-A700 (eBioscience), anti-CD4-PE/Cy7 (BioLegend, San Diego, CA, USA), anti-CD8-BrilliantViolet605 (BioLegend), anti-CD11b-PE/Cy5 (eBioscience), anti-Ly6C-APC/Cy7 (BioLegend), anti-Ly6G-FITC (BioLegend), anti-F4/80-PE (eBioscience), anti-CD64-APC (BioLegend), and 7-AAD (BioLegend). Purified anti-FcɣRIII/II (BioLegend) was used to block Fc-receptors. Data were acquired on LSR II flow cytometer (BD) and analyzed with FlowJo software v10.0 (BD).

Bladders were digested at 37°C for 30 minutes in RPMI-1640 with 10mM HEPES, collagenase D, and DNAse (all Sigma-Aldrich), forced through a 70 μm cell strainer (Corning), and washed with 5% fetal bovine serum (FBS, ThermoFisher Scientific) in PBS. Urines were diluted in fluorescence-activated single cell sorting (FACS) Buffer (PBS supplemented with 5 mM EDTA and 3% FBS) for 30 minutes (pH neutralization), prior to staining. Single cell suspensions resuspended on ice-cold FACS Buffer were stained with anti-CD45 eFluor450 (eBioscience), anti-CD3-A700 (eBioscience), anti-CD4-PE/Cy7 (BioLegend), anti-CD8-BrilliantViolet605 (BioLegend), anti-CD11b-PE/Cy5 (eBioscience), anti-Ly6C-APC/Cy7 (BioLegend), anti-Ly6G-FITC (BioLegend), anti-F4/80-PE (eBioscience), anti-CD64-APC (BioLegend) and 7-AAD (BioLegend). Purified anti-FcɣRIII/II (BioLegend) was used to block Fc-receptors. Data was acquired on LSR II flow cytometer (BD) and analyzed with FlowJo software v10.0 (BD).

Flow cytometric analysis of white blood cells (WBC) isolated from C57BL/6NJ was performed, as described previously (Paul et al., 2017 (link)). Briefly, peripheral blood was collected and centrifuged at 2000×g for 15 min and plasma removed. Red blood cell lysis buffer at 1X dilution (eBioscience) was added and cells were incubated for 10 min at room temperature on an orbital shaker followed by addition of 1X Phosphate Buffered Saline Solution (PBS, Thermo Fisher Scientific). Cells were then centrifuged at 500×g for 5 min at 4°C to pellet out the WBC, fixed in 2% paraformaldehyde (Thermo Fisher Scientific) for 15 min on ice, washed, and incubated with Fc block (CD16/32 Block) for 20 min, followed by probing with anti-CD45-FITC, anti-CD4-PE, anti-CD8a-PerCP, anti-CD11b-PECy5, and anti-Ly6G-PE antibodies (all purchased from Thermo Fisher Scientific) for 1 h at room temperature in the dark. Samples were then washed twice in 1X PBS, and acquired using a Guava Flow Cytometer (Millipore). Unstained and single stained compensation controls were used during acquisition and FlowJo software (version 10.3.0) utilized for cytometric analysis.