Dri chem 3500

After rat blood was collected at the end of the experiment, serum ALP levels were assessed using a blood biochemical analyzer (DRI-CHEM 3500 s; Fujifilm, Kanagawa, Japan). Serum osteocalcin levels were determined using a rat osteocalcin ELISA Kit (LifeSpan BioSciences, Seattle, WA). Serum PICP and NTx levels are the markers of bone formation and bone resorption respectively. PICP was measured by using PICP ELISA kit (Cloud-Clone Corp., Houston, TX). On the other hand, NTx was measured by using NTx ELISA kit (Cloud-Clone Corp., Houston, TX).

Organ dysfunction and injury were assessed using a blood biochemical analyzer (DRI-CHEM 3500s; Fujifilm, Kanagawa, Japan) that measured serum levels of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), total billirubin (TBIL), creatine-phospho-kinase (CPK), creatine phosphokinase-MB (CKMB), lactic dehydrogenase (LDH), creatinine (CRE), and blood urea nitrogen (BUN).

The Cisd2 BAC transgenic (Cisd2TG) mice were generated on a C57BL/6 background (Shen et al., 2017 (link); Wu et al., 2012 (link)). The Cisd2TG mice carry four copies of Cisd2 genes, namely two endogenous Cisd2 alleles and two transgenic copies of the Cisd2 BAC. Since the prevalence of NAFLD in males is significantly higher than in females at all ages (Lonardo et al., 2019 (link)), male mice were used for all experiments in this study. The mice were maintained at 21 ± 1°C under a regular 12/12 h light/dark cycle and fed a normal chow diet ad libitum. When assessing age‐dependent hepatic steatosis, the experiments were performed using wild‐type mice aged 3 and 26 months old. Additionally, Cisd2TG mice were sacrificed at 26 months old. The body weights and liver weights were recorded at sacrifice. The serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) of the mice were determined using a DRI‐CHEM 3500s (FUJIFILM). All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of National Yang Ming Chiao Tung University (No. 1080410).

C57BL/6 mice were anesthetized with rumpun (10 mg/kg) and ketamine (100 mg/kg) and intradermally injected with PBS, CCNVs or SCCNVs (20 μg of nanovesicles in 50 μl of PBS). Blood samples were obtained at various time points. Serum was obtained from the blood samples by centrifugation at 3000 × g for 30 min. The levels of AST, ALT, creatinine and BUN in the serum were determined with a DRI-CHEM 3500 S chemical analyzer (Fujifilm, Japan). For histological analysis, major organs (the liver, lungs, spleen, heart and kidneys) were harvested 14 days after the first injection. The tissues were fixed in 4% PFA overnight at 4 °C and dehydrated in a 30% sucrose solution. The tissues were embedded in OCT compound (Scigen Scientific) and sectioned at a thickness of 10 μm using a cryostat microtome (Leica, Germany). The sections were stained with hematoxylin (Cancer Diagnostics, NC, USA) and eosin (BBC Biochemical, WA, USA) and imaged using an optical microscope (Olympus, Tokyo, Japan).

Liver function was determined on the basis of the enzymatic analysis of glutamate oxaloacetate transaminase/aspartate transaminase (GOT/AST) and glutamate pyruvate transaminase/alanine aminotransferase (GPT/ALT) [30 (link)]. GOT and GPT activities were assayed using a biochemical blood analyzer (DRI-CHEM 3500s, Fujifilm, Japan).