Human il 10

Fasting serum samples were used for biochemical analyses, including TG, CHOL, LDL, HDL, CHOL, AST, ALT, insulin, C-peptide, free fatty acid, BUN, CRE, eGFR, HbA1c, and glucose analyses, at Union Clinical Laboratory (Taiwan, TAF No. L1447-150325/CAP No. 6979606). The cytokine profiles were determined by enzyme-linked immunosorbent assay (ELISA) kits and performed at the Department of Life Sciences (Institute of Biomedical Science, National Chung Hsing University, Taichung, Taiwan). The ELISA kits included human IL-6 (Cat# 900-K16, PeproTech, USA), human IL-10 (Cat# 900-K21, PeproTech), human IL-17A (Cat# 900-K84, PeproTech), human TNF-α (Cat# 50-114-2609, eBioscience, USA), and human IL-1β (Cat# 437005, Biolegend, USA). A glutathione peroxidase assay kit (Cat# 703102, CAYMAN, USA) and superoxide dismutase assay kit (Cat# 706002, CAYMAN) were used in this study according to the manufacturers’ standard protocol.

Tissue sections were deparaffinized in xylenes and rehydrated. Antigen retrieval was performed with 10 mmol/L citrate buffer (pH 6.0) and sections were blocked with SuperBlock (ThermoFisher Scientific, Waltham, MA). Primary antibodies against human mitochondria (1:200), murine KIM‐1 (1:500) (Abcam) or human IL‐10 (1:200) (eBioscience) were incubated on sections overnight at 4°C. For human mitochondria, an Alexa Fluor 647‐labelled secondary antibody was hybridized on sections for 2 hours at room temperature. We have previously characterized this antibody to specifically detect human MSC in kidneys.21 For KIM‐1, a horseradish peroxidase conjugated secondary antibody and reacted with 3,3′‐diaminobenzidine (DAB). Slides were coverslipped using ProLong Diamond anti‐fade mounting medium containing 2‐(4‐amidinophenyl)‐1H‐indole‐6‐carboxamidine (DAPI) (Life Technologies, Carlsbad, CA) or Permount mounting medium. Control sections were stained with species‐specific IgG as isotype controls. Cell death was measured using an In Situ Cell Death Detection, Fluorescein kit (Roche, Basel, Switzerland). Examples of KIM‐1 and TUNEL staining in healthy mice are shown in Figure S2.

Tissue culture supernatant was collected from LPS- or galectin-1-matured dendritic cell cultures after 24 h of maturation and stored at −80 °C until analysis. Cytokine secretion was determined using human IL-10 (eBioscience) and IL-12 (BD Bioscience) sandwich ELISA kits.

After 5 days incubation, the culture supernatants were aspirated and assayed for concentrations of the cytokines IFNγ, IL4 and IL10, TGFβ1 (latent form) using the following ELISA kits; Human IFNγ (no. 88–731); Human IL4 (no. 88–7046); Human IL10 (no. 88–7106); and Human LAP (TGF β1) (no. 88–50390), (all from eBiosciences), as per the manufacturer’s instructions. Mean values of triplicate measurements were tabulated.

Human and mouse cytokines were measured in kidney homogenates using species‐specific enzyme‐linked immunosorbent assays (ELISA) for human IL‐10 (Cat# D1000B) human indoleamine 2,3‐dioxygenase (IDO; Cat# DY6030‐05), murine IL‐10 (Cat# M1000B) and murine IFNγ (Cat# MIF00) (R&D Systems, Minneapolis, MN). Antibodies were demonstrated by the manufacturer to be free of interspecies cross‐reactivity (ie. the human and mouse IL‐10 antibodies detected only IL‐10 from that species). ELISAs were loaded with tissue homogenates at a concentration of 2 mg/mL and developed according to manufacturer protocols. Western blotting was performed by loading 20 μg of total protein were resolved by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis on a 4%‐12% polyacrylamide gel under reduced conditions. Proteins were transferred onto nitrocellulose membranes, blocked in 5% bovine serum albumin and hybridized with primary antibodies overnight at 4°C. Primary antibodies (dilutions in parentheses) included human IL‐10 (1:500), murine IL‐10 (1:500) (eBioscience, San Diego, CA), or β‐actin (1:5000) (Abcam, Cambridge, MA). Species‐appropriate and fluorescently labelled secondary antibodies (dilutions ranging from 1:1000 to 1:10000) (Abcam) were incubated for 1 hour at room temperature. Blots were imaged using a ChemiDoc MP system (Bio‐Rad, Hercules, CA).