Blenzyme 4

Single cells were isolated and analysed by flow cytometry as described previously^{34 (link), 35 (link)}. Briefly, mouse kidney and spleen were digested with blenzyme 4 (0.1 mg/ml, Roche Inc., Indianapolis, Indiana, USA) and DNase I (100 U/ml) into cell suspension. Then lymphocytes were separated by discontinuous density centrifugation on Percoll gradients (40%, 60%, and 80%, Sigma Aldrich, St. Louis, Missouri, USA). After being stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors, eBioscience, San Diego, California, USA), the isolated cells were fixed and permeablised with IC Fixation Buffer and Permeabilization Buffer (eBioscience). Subsequently, these cells were stained with FITC-conjugated anti-mouse CD4, APC-conjugated anti-mouse IFN-γ, IL-4-APC, IL-17-APC, CD25-APC, or PE-conjugated Foxp3. Flow cytometry was performed on a FACS Calibar using the CellQuest Pro Analysis software (BD Biosciences, Franklin Lakes, New Jersey, USA).

Graft heart and spleen tissues were digested by blenzyme 4 (Roche Inc, Indianapolis, IN, USA) into cell suspension. The white blood cells were then enriched by centrifugation through discontinuous Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) density gradients (40%, 60% and 80%). After being stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors, eBioscience, San Diego, CA, USA) for 12 hours in CO₂ incubator, the gathered cells were fixed for 30 minutes in IC Fixation Buffer (eBioscience) and permeablized by 1x Permeabilization Buffer (eBioscience) for 30 minutes. Then cells were stained with fluorescein isothiocyanate (FITC) -conjugated anti-mouse CD4, allophycocyanin (APC) -conjugated IFN-γ, phycoerythrin (PE) -conjugated IL-4, Foxp3-APC or IL-17A-PE (eBioscience) for overnight at 4°C. After being extensively washed, single cells were analyzed by FACSCaibur flowcytometer (BD Biosciences, San Jose, CA).

Mouse kidney was digested into a single cell suspension with Blenzyme 4 (Roche, Indianapolis, IN, USA) at 37 °C for 30 minutes followed by sieving. Then cells from the mouse kidney or peripheral blood of health or AKI patients were fixed for 30 min in IC Fixation Buffer (eBioscience), permeabilized in permeabilization buffer (eBioscience) for 15 min at room temperature, and then labeled with the combinations of the following antibodies including APC-conjugated iNOS (eBioscience), FITC-F4/80 (BioLegend) or ERM1 (recognized human macrophage F4/80 antigen, Santa Cru), APC-Mincle (Santa Crus), PE-NPY (Santa Cruz) for overnight at 4 °C. After being washed, cells were gated and analyzed by the FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).