Glycerol assay kit

Serum was isolated from peripheral blood by centrifugation and stored at −80 °C until analysis. Cell supernatant was collected after the cells were cultured with basal condition or with isoprenaline stimulation for 24 h and immediately analyzed. Adipose tissue was freshly collected and processed immediately as previously described [36 (link)]. A Free Fatty Acid Assay Kit (Nanjing Jiancheng, A042-2) and a Glycerol Assay Kit (Nanjing Jiancheng, F005-1) were used to measure FFA and glycerol levels according to the manufacturer’s instructions.

The levels of NEFAs in the serum were measured using an automatic biochemical analyser (Roche, cobas c311). Levels of glycerol were determined by a Glycerol Assay Kit (Nanjing Jiancheng Bioengineering Institute, F005-1-1). Levels of TNF-α, IFN-γ, IL-1β and IL-6 in adipose tissue lysate were assessed by ELISA (eBioscience, ProcartaPlex multiple cytokines ELISA kit, PPX-06).

The epididymal adipose tissue was dissected, the fresh adipose tissue was weighed and rapidly rinsed with PBS. The adipose tissue was cut and incubated with isoproterenol (10 μM) for 2 h, and homogenized in a cold cleavage buffer to prepare the adipose tissue homogenate. Then the homogenate was centrifuged at 12,000 × g at 4 °C for 15 min, and the supernatant was collected. For 3T3-L1 adipocytes, the adipocytes were treated with or without 50 ng/ml TNF-α or 3 μg IKKε plasmid or 4 μM siIKKε for 24 h and then treated with or without 10 μM ISO or 10 μM BJ for 24 h. Then the cells were lysed in a petri dish, scraped off, and centrifuged at 12,000 × g at 4 °C for 15 min before the supernatant was collected.
Glycerol or cAMP content in adipose tissue and 3T3-L1 adipocytes was determined by a glycerol assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) or a mouse cAMP assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). And concentrations were further standardized according to protein concentration.

The treated cells were washed once with PBS and were then incubated with DMEM (no phenol red) (Gibco, USA) with or without GST-irisin for 4 h. Samples of the media were collected and assayed for glycerol levels using a glycerol assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Serum levels of non‐esterified fatty acid (NEFA) and glycerol were measured using NEFA kit (A042, Jiancheng Biotechnology) and Glycerol Assay kit (F005‐1, Jiancheng Biotechnology), respectively. These assays were performed according to manufacturer's instructions. Serum levels of triglyceride (TG), total cholesterol (TC), high‐density lipoprotein cholesterol (HDL‐C), and low‐density lipoprotein cholesterol (LDL‐C) were measured by an automatic biochemical analyzer (Chemray 240, Rayto Life and Analytical Sciences).
Serum alanine (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) microplate test kits were obtained from Nanjing Jiancheng Bioengineering Institute. These assays were performed as previously described (Wang et al., 2020 (link)). Briefly, ALT, AST, and ALP activities were evaluated at 37°C for 15 min by assessing for a decrease in absorbance at a wavelength of 510 nm, with Chemi Lab ALT, AST, and ALP assay kits, respectively.

Dulbecco’s modified Eagle’s medium (DMEM)/high glucose was obtained from Biological Industries (Israel). Calf serum (CS) for cell culture was purchased from GIBCO BRL (Grand Island, NY, USA). Penicillin/streptomycin (P/S) and fetal bovine serum (FBS) were obtained from Biological Industries (Israel). Rosiglitazone (Rosi), dimethyl sulfoxide (DMSO) and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma-Aldrich (St Louis, MO, USA). Insulin was from Roche (Switzerland), and dexamethasone (DEX) was from Adamas (Switzerland). The 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) and cell lysis buffer were from Beyotime (Shanghai, China). Compound C was purchased from Calbiochem (San Diego, CA). Bovine serum albumin (BSA) was from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). The antibodies against AMPK, phospho-AMPK-α (Thr172), FABP4, C/EBPβ, C/EBPα and PPARγ were from Cell Signaling Technology (Beverly, MA, USA), β-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Triglycerides Kit, Glycerol Assay Kit and Non-esterified fatty acids Kit were purchased from Nanjing Jiancheng Bioengineering Institute. Mouse Adiponectin ELISA Kit was purchased from MULTI SCIENCES.

Changes in intracellular osmotic pressure were determined by measuring glycerol content using the Glycerol Assay Kit (Jiancheng, Nanjing, China), according to the provided protocol. Protein concentrations were determined using the Bradford reagent (Beyotime, Shanghai, China) with BSA employed as a standard. The concentrations of glycerol were calculated as the values of the content of glycerol divided by the content of protein. Results are the mean of triplicate experiments ± SDs.