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.
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.