To characterize immune infiltration, the entire UUO kidney and its corresponding contralateral control were minced and then digested in 2 ml of collagenase (400 U of type I collagenase) in Dulbecco’s modified Eagle’s medium at 37°C for 25 min. Spleen and lymph node cells were ground into single-cell suspensions and filtered through 100-nm mesh before immunostaining cells with a fixable viability dye. For fluorescence-activated cell sorting (FACS) analysis, phycoerythrin (PE)–conjugated anti–LYVE-1 antibody was obtained from R&D Systems. The following additional antibodies were obtained from BioLegend: allophycocyanin (APC)/Cy7-conjugated anti-mouse CD45 antibody, BV510-conjugated CD45 antibody, fluorescein isothiocyanate (FITC)–conjugated anti-mouse/human CD11b antibody, PE-conjugated anti-mouse CD8 antibody, peridin chlorophyll protein (PerCP)/Cy5.5-conjugated anti-mouse CD8 antibody, APC-conjugated anti-mouse Ly6G antibody, FITC-conjugated anti-mouse F4/80 antibody, PE-conjugated anti-mouse Ly6G antibody, PE-conjugated anti-CD11c antibody, APC-conjugated anti-mouse CD3 antibody, PerCP/Cy5.5-conjugated anti-mouse/human CD3 antibody, PerCP/Cy5.5-conjugated anti-mouse I-AD antibody, APC-conjugated anti-mouse CD4 antibody, Pacific blue–conjugated anti-mouse CD8 antibody, FITC-conjugated B220Ab, BV421-conjugated anti-mouse CD86 antibody, APC-conjugated anti-mouse CCR7 antibody, Alexa Fluor 488–conjugated anti-mouse CCR7 antibody, PerCP/Cy5.5-conjugated anti-mouse CD19 antibody, PE-conjugated anti-mouse NK1.1 antibody, FITC-conjugated anti-mouse CD44 antibody, and PE-conjugated anti-mouse LY6C. Proliferating cells were analyzed by flow cytometry using an anti-Ki67 antibody (clone SolA15, eBioscience) with the Transcription Factor Staining Buffer Set (00-5523-00, eBioscience). The dead cells were excluded by staining with APC/Cy7-conjugated Zoom. APC/Cy7-conjugated isotype, FITC-conjugated isotype, PerCP/Cy5.5-conjugated isotype, PE-conjugated isotype, APC-conjugated isotype, Alexa Fluor 488–conjugated isotype, and BV421-conjugated isotype were used as isotype controls. Positive cells were sorted using a BD FACS, and the data were analyzed using FlowJo v7.6.3 software.

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