Apc conjugated anti f4 80

For tissue preparation, the obstructed kidney was minced and incubated with 2mg/mL collagenase II (Sigma), 1mL DMEM, and DNAse (BioRad 10ul/mL) in a shaker at 37C for 1 hour. The tissue was filtered (70 μm), centrifuged, and incubated in red blood cell lysis buffer (15.5mM NH₄Cl, 1mM KHCO₃, 10 μM EDTA) at 37C for 5 minutes, neutralized with PBS, centrifuged, and resuspended in PBS with 1%FBS. The tissue was passed through a 50 μM strainer, and tissue from COL-Cre mice (and controls) was passed through a 24 μM filter to remove the glomeruli. The FACSAria II from BD Biosciences in the Research Flow Cytometry Core Laboratory at the Nashville VA Medical Center was used for sorting of GFP+ cells.
For flow cytometry, samples were incubated with Fc blocking antibody (Biolegend) on ice, then incubated with APC-conjugated anti-F4/80, anti-CD140b (PDGFRβ), or the appropriate APC-conjugated isotype controls (Biolegend) for 30 minutes at room temperature. Cells were analyzed on a FACS Canto II cytometer with FACSDiVa v6.1 software for data acquisition and analysis at the Nashville VA Medical Center’s Flow Cytometry Laboratory.

To collect samples for flow cytometry analysis, mice were euthanized via CO2 asphyxiation. Peripheral blood was collected via cardiac puncture. Erythrocytes within blood were lysed with ammonium chloride (StemCell Technologies) and the remaining leukocytes were isolated for flow cytometry analysis. Bone marrow was collected via centrifugation (1000g for 5 min) of isolated tibiae. The dorsal tissue was excised and digested with collagenase type 1-A (1 mg/ml, Sigma) at 37 °C for 30 min and further separated with a cell strainer to create a single cell suspension. Single cell suspensions of tissues, blood, and bone marrow were stained for flow cytometry analysis using standard methods and analyzed on a FACS-AriaIIIu flow cytometer (BD Biosciences). The antibodies used for identifying cell populations of interest were: PerCP-Cy5.5 conjugated anti-CD45 (BioLegend), APC-Cy7 conjugated anti-CD11b (BioLegend), BV421 conjugated anti-CD11b (BioLegend), APC conjugated anti-Ly6C (BioLegend), BV510 conjugated anti-Ly6C (BioLegend), APC-Cy7 conjugated anti-Ly-6G (BioLegend), PE-Cy7 conjugated anti-GR-1 (BioLegend), APC conjugated anti-F4/80 (BioLegend), PE-Cy7 conjugated anti-CD206 (BioLegend), AlexaFluor488 conjugated anti-CD86 (BioLegend), PE conjugated anti-CD49d (BioLegend).