Multifunctional microplate reader

Each experimental group contained five samples consisting of six zebrafish. Ice water was used for euthanasia, and the samples used for biochemical tests were crushed via ultrasonic soundwaves. Triglycerides (TG), total cholesterol (TC), malondialdehyde (MDA), and GSH-px levels were measured with commercial assay kits (Jiancheng, Nanjing, China) according to the manufacturers’ instructions. The cell biochemical measurements were all carried out following the manufacturers’ instructions. The quantitation results of the above kits were read with a multifunctional microplate reader (BioTek, Winooski, VT, USA).

The concentration of soluble sugar in fruit was measured as described by Liu et al. (2018) (link). The sample of apple flesh was placed in a test tube, then 5 ml of distilled water was added and mixed after cutting it into chunks. The supernatant was collected after 30 min of boiling water bath. This step was repeated twice, using distilled water to adjust the volume of the solution to 10 ml. The absorbance of the solution was measured at 630 nm after adding sulfuric acid and anthrone. Six biological replicates and three technical repetitions were conducted for each treatment.
The concentration of anthocyanin was measured as described by Zheng et al. (2021) (link). The sample of fruit peel was ground with liquid nitrogen rapidly and then extracted with 1.5 ml of 1% (v/v) HCl methanol at 3°C for 30 h in darkness. The concentration of anthocyanin was measured by a multifunctional microplate reader (BioTek, USA). Six biological replicates and three technical repetitions were conducted for each treatment.
The concentration of titratable acid was measured by NaOH titration (Nie et al., 2012 (link)), and the fruit hardness was measured by a HP-230 hardness tester. The diameter of fruit was measured with a vernier caliper. Single fruit weight was measured by 1% precision electronic balance, and the average of 30 measured values was taken.

Cell viability was measured using a Cell Counting Kit-8 (CCK-8) kit (Biosharp, China). Cells were seeded into the 96-well plate (5×10³ cells/per well). After cell attachment, cells were treated with different concentrations of OSI or other compounds for 72 h. After that, the original medium was removed, and a new culture medium with 10% CCK-8 was added to each well. The cells were incubated for 2.5 h. Then the absorbance of each well was measured at 450 nm using a multifunctional microplate reader (Bio-Tek, United States).

The Limulus amebocyte extract kit (catalog no. CE80545, Biondo, Xiamen, China) was used to measure the plasma concentration of LPS. The plasma LPS concentration was measured by dilution in a sample processing buffer at a 1:10 ratio and heated for 10 min at 70 °C. The concentration of total bile acid in serum was measured by the total bile acid kit (catalog no. E003-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Briefly, serum samples and reagents were added to 96-well plates and then incubated at 37 °C, after which the optical density (OD) was measured at 490 nm using a multifunctional microplate reader (BioTek, Winooski, VT, USA).