Multifunctional microplate reader

The cell viability was determined by MTT assay as described previously [17 (link)]. Cells were seeded at 2 × 10⁴ cells/well into 96-well culture plates. After being incubated for 12 h, the cells were treated with SGGY (0.1, 0.5, 1, or 2 mg/mL) for 4, 8, or 12 h and subsequently stimulated by H₂O₂ (300, 600, 900, 1200, or 1500 μM) for 2 h. The cells of the control group were cultured in the same serum-free medium but without SGGY and H₂O₂ supplementation. After removing the medium with H₂O₂, the cells were rinsed twice with phosphate buffer solution (PBS). The MTT solution was added (final concentration 0.5 mg/mL) and the cells continued to be incubated at 37 °C for 4 h. Next, the supernatant in each well was removed and the formazan crystals formed by live cells were dissolved in each well by adding 150 μL of DMSO and shaking gently for 20 min. Absorbance of each well was determined at 490 nm by a multifunctional microplate reader (PerkinElmer Co., Boston, MA, USA). The cell viability was displayed as the percentage of the control group.

Cell viability was assessed with Cell Counting Kit‐8 (CCK‐8) (Sangon Biotech (Shanghai) Co., Ltd.). To determine cell viability, Ins‐1 cells were seeded at a density of 5000/well in 96‐well plates (100 μl/well). Then, the cells were cultured overnight and treated with culture medium containing 10⁻⁵‐10^‐9 mol/L LB for different times. The absorbance values were measured at 450 nm using multifunctional microplate reader (PerkinElmer, Waltham). The experiment was repeated 3 times. $\mathbf{C}\mathbf{e}\mathbf{l}\mathbf{l}\mathbf{v}\mathbf{i}\mathbf{a}\mathbf{b}\mathbf{i}\mathbf{l}\mathbf{i}\mathbf{t}\mathbf{y}\left(\%\right)=\frac{\mathbf{O}\mathbf{D}\mathbf{S}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}‐\mathbf{O}\mathbf{D}\mathbf{B}\mathbf{l}\mathbf{a}\mathbf{n}\mathbf{k}}{\mathbf{O}\mathbf{D}\mathbf{S}\mathbf{a}\mathbf{m}\mathbf{p}\mathbf{l}\mathbf{e}‐\mathbf{O}\mathbf{D}\mathbf{B}\mathbf{l}\mathbf{a}\mathbf{n}\mathbf{k}}\times 100\%$

After drug treatment for 24 h, the culture medium of each group was harvested to detect levels of TNFα and IL-1β via ELISA (Boster, China). A multifunctional microplate reader (PerkinElmer, USA) was used to measure the OD values of each group. TNFα and IL-1β contents were calculated using a standard curve according to manufacturer’s instructions. The experiment was performed as described previously [20 (link)].

Intracellular acetyl‐CoA levels were measured by an acetyl‐CoA assay kit (Comin, Bio). The cells were seeded in six‐well plates at 1 × 105 cells (SW480) or 2 × 105 cells (HT29) per well, cultured under normal medium overnight, and then left in specific treatment at the 48 or 24 h time‐point before the subsequent test. The cells were harvested in 200 µL lysis buffer according to the instructions. Then, 184 µL acetyl‐CoA detection solution was added to the wells of a 96‐well plate and placed at room temperature for 5 min. Then, 20 µL of sample was added, and chemiluminescence was immediately measured by a multifunctional microplate reader (PerkinElmer). The values were recorded every 60 s, and the relative concentration (y) of intracellular acetyl‐CoA was calculated according to the maximum ΔA (ΔA = A₂−A₁, y = 1640*ΔA + 0.012). The protein concentration of each sample was detected by the BCA protein assay kit.

All of the fluorometric experiments were recorded on a multifunctional microplate reader (Perkin-Elmer Corporation USA). The stock solutions of BSA and the tested components were diluted to the required experimental sample concentrations and mixed in multiple colorimetric tubes. For SaB and SYR, the range of concentrations was 0, 2.5, 5, 10, 15, 20, 25, 30, 35, 40 μM from 1 to 10; for HSYR, the range of concentrations was 0, 25, 50, 60, 70, 80, 90, 100 μM from 1 to 8; for DSS, CYT, URI, ADE, and Phe, the range of concentrations was 0, 20, 40, 50, 60, 70, 90, 100 μM from 1 to 8. The test solutions for fluorescence spectroscopy were prepared with different concentrations via stepwise dilution. To avoid the inner filter effect, we used very dilute solutions in the experiment, ensuring a final concentration of BSA of 5 μM. The test solutions were incubated at 310 K for 40 min and then transferred into 96-well black fluoro-microplates (CORNING, USA); the fluorescence spectroscopy tests were carried out at the same temperature to simulate the body temperature. The excitation wavelength was 280 nm, and the emission spectra were recorded from 300 to 500 nm. The maximum emission intensities were used to calculate binding parameters. In order to correct the background, appropriate blanks were subtracted according to series preliminary experiment.

ATP content was measured by luminometric assay, based on luciferin-luciferase reactions (Drew and Leeuwenburgh, 2003 (link)), using the Beyotime chemical luciferase ATP assay kit. Briefly, mitochondrial membranes were lysed in a buffer that contained 10 mmol/L Tris and 0.05% Triton X-100. Then, 50 μL of this lysate was added, and the luciferin-luciferase assay mixture was transferred to a white microplate. The results were measured on a multifunctional microplate reader (Perkinelmer, United States).

NO in the serum was detected by using NO assay kit (Nanjing Jiancheng Bioengineering Institute, China). All the operations in the experiment were carried out in accordance with the specification. The OD values were read by the multifunctional microplate reader at 550 nm (PerkinElmer, USA), and then the result was calculated according to the formula given by the specification.