Ldh assay kit

Cells were seeded into 6-well plates and incubated as described in Section 4.3.9, and then the activities and levels were measured by SOD, CAT, MDA, and LDH assay kits (Beyotime Biotechnology, Shanghai, China). The assay was evaluated in five copies and repeated three times.

Lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK‑MB) were regarded as biomarkers released by cells subjected to myocardial ischemia. We detected the levels of LDH and CK-MB to measure the degree of AC16 cell damaged by HR treatment. LDH levels were detected using LDH assay kits (#C0016, Beyotime, Shanghai, China) following the manufacturer’s instructions. CK-MB levels were evaluated by ELISA kits (#EHCKMB, Invitrogen, CA, USA), and the absorbance was measured at 450 nm using a microporous plate reader.

The supernatant of AML12 was collected and the activity of LDH leakage was detected according to the instructions of LDH assay kit (Beyotime, Shanghai, China). Cells were then washed with PBS and the triglyceride content in each group was measured according to the instructions of TG assay kit (Jiancheng, Nanjing, China).

For the MTT assay, HUVECs (5 × 10³ cells/well) were seeded in 96-well plates, cultured overnight and treated with varying concentrations of TMAO (0, 10, 50, 100 μmol/l) for 24 h. And then added 10 μl MTT solution to each well at a final concentration of 0.5 mg/ml and incubated for an additional 4 h. At the end of incubation, DMSO (150 μl) was added to each well and then the absorbance was measured at 570 nm by a microplate reader (Varioskan Flash, Thermo Fisher).
HUVECs were incubated with indicated concentrations of TMAO for 24 h, lactate dehydrogenase (LDH) activity in the culture media and cell lysates was respectively analysed using an LDH Assay Kit (Beyotime Institute of Biotechnology, China). Results were expressed as the percentage of LDH leakage, which was the ratio of LDH activity in the media to total LDH activity.
Cell proliferation was analysed using the 5-bromo-2-deoxyuridine (BrdU) assay. Briefly, equal numbers (5 × 10³ cells/well) of HUVECs were seeded in 96-well plates. Confluent HUVECs were then treated with TMAO (0, 10, 20, 40, 80, 100, and 200 μmol/l). Cell proliferation was measured for 48 h using BrdU assay kit according to the manufacturer’s protocol with stop solution (Roche Cell Proliferation ELISA BrdU colorimetric assay). The absorbance at 450 nm was measured within 5 min after adding the stop solution [23 (link)].

LDH assay kit (REF: C0016, Beyotime, China) was used to detect LDH activity referring to its instruction. The absorbance of each well at 450 nm is detected.