F4 80 percp

Kidneys were collected from sacrificed animals for flow cytometry analysis following the standard manufacturer's protocol. Briefly, kidneys were reperfused, excised, and incubated in collagenase IV for 30 min. Then, monocytes were separated by Percoll (Sigma, St. Louis) gradient. We analyzed the renal cells by multicolor flow cytometry. The monoclonal antibodies used were F4/80 PerCP, CD11b PE, CD206 FITC, MIG PE, IL10 APC, p40 (IL12/IL23) PE‐Cy7, CCR7 PerCP‐Cy5.5, IFNγ FITC, CD45.1 Pacific Blue, CD45.2 APC‐Cy7, GM‐CSF PE, MHC II IAB PerCP, and CD36 FITC (all purchased from BD Biosciences, Franklin Lakes). Samples were acquired on a FACSCanto using FACSDIVA software (BD Biosciences), followed by analysis with FLOWJO software (Tree Star, San Carlo, CA). Fluorescence voltages were determined using matched unstained cells. Compensation was performed using cells (BD Biosciences) single‐stained with CD3 PErCP, CD4 FITC, CD8 APC‐CY7, CD4 PE‐CY7, CD4 PerCP‐Cy5.5, CD3 PE, or CD3 APC. Samples were acquired up to at least 200,000 events in a live mononuclear gate, followed by a doublets exclusion gate, following the standard manufacturer's procedure. The compensation process was performed according to the “fluorescence minus one” method.

After 24 h incubation, LM were detached (PBS, 4 °C), centrifuged and re-plated (U bottom plate, 96 wells) for subsequent labeling with a conjugated antibody mix: F4/80-PerCP, CD11b-APC, CD11c-PECy7, CD86-FitC and CD206-PE (1: 100) (BD Biosciences, Franklin Lakes, NJ, USA). Beads incubated with each antibody were used as control compensators. Samples were analyzed on a FACSCanto II cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using Diva-SofwareTM for data acquisition. 50,000 events per sample were acquired and FlowJo 10.0.7 software was used for data analysis.