Isolation of Renal Mononuclear Cells

Intravascular antibody injection of anti-CD45.1 (clone A20, eBioscience, 12-0453-82) was administered to mice 3 min before euthanasia, allowing us to distinguish intravascular and tissue-infiltrating cells as previously described (51 (link)). After organ harvest and removal of renal capsule, renal tissues were crushed into small pieces and incubated in RPMI 1640 with 10% fetal bovine serum, collagenase D (100 IU/ml) with CaCl₂ (2 mM), and MgCl₂ (2 mM) at 37°C for 45 min. After enzymatic digestion, gentleMACS was used to disrupt remaining tissue. Cell suspensions were filtered through 70-μm strainer and resuspended in phosphate-buffered saline (PBS). Mononuclear cells were isolated after passing the single cell suspension through Ficoll density gradient centrifugation.