Quantification of kidney cell populations by flow cytometry.

Multiparametric flow cytometry was used for the identification of the macrophage and epithelial cell population. Kidneys were diced, incubated at 37°C for 30 minutes with 0.5 mg/mL Liberase DL (Roche Basel) and 100 U/mL DNase (Roche Basel) in serum-free DMEM, and filtered (40 μm). For staining, 10⁶ cells were dissolved in 50 μL of buffer and preincubated with 0.25 μL CD16/CD32 (BioLegend). Next, 3 μL of DAPI (1:15000) (Sigma-Aldrich) was added to stain dead cells. Cell suspensions were incubated with specific fluorochrome-conjugated antibodies (BioLegend) listed in Supplemental Table 5. For each experiment, flow minus one (FMO) controls were performed for each fluorophore to establish gates by using corresponding antibodies listed in Supplemental Table 5. Fluorescence intensity was measured in a FACSCanto II cytometer (BD Biosciences) and analyzed with FlowJo 10.2 software. For each kidney sample, at least 20,000 singlets were analyzed in triplicates. Cell gating was initially based in the forward versus side scatter (FSC vs. SSC) plot. Next, dead cells were excluded. Identification of the macrophage cell population was based on the presence of CD45, expressed in inflammatory and hematopoietic cells and F4/80, a specific macrophage surface marker. CD86 and CD206 were used to determine M1 and M2 macrophage subpopulations, respectively (38). Identification of the epithelial cell population was based on the presence of the epithelial cell adhesion molecule (EpCAM) and the absence of CD45. The subsequent display of positive CD24 determined injured proximal tubule epithelia (39). Numbers in quadrants indicate cell proportions in the percentage of cells that coexpress both markers.