Atp assay bioluminescence detection kit

Whole blood from normotensive and hypertensive mice was collected at different time points. Plasma was prepared by spinning the whole blood at 1500g for 15 min and collecting the supernatant. ATP levels were determined with an ATP assay bioluminescence detection kit (Beyotime). For testing tissue ATP release, aorta and renal artery were removed from PBS-perfused mice and cultured in 30 μl of RPMI 1640 for 3 hours. To measure ATP production by blood cells, 50 μl of whole blood was centrifuged at 300g for 5 min to remove plasma. Blood cells were resuspended in 50 μl of RPMI 1640 and transferred into a fresh well for a 3-hour incubation. After 3 hours, the culture plates containing blood cells or tissue were centrifuged at 300g for 5 min and supernatants were then collected for ATP measurement.

Zhejiang Hospital and the Fourth Affiliated Hospital of Zhejiang University reviewed and approved the human studies. All the participants signed a written informed consent form before being enrolled in the study. Patients with a history of hypertension between 40 and 70 years of age and normotensive controls were recruited. Their BP was measured on two consecutive days. Patients were assigned to the HTN group when either average SBP was ≥140 mmHg or average DBP was ≥90 mmHg. Hypertensive patients (by history) who were taking anti-hypertensive medications and had both average SBP below 140 mmHg and average DBP below 90 mmHg were assigned to the C-HTN group. The NTN group is normotensive controls with normal measured BP and no previous history of hypertension. Patients with histories of autoimmune diseases, lung diseases, asthma, stroke, AIDS, cancer, and transplantation and those having symptoms or history of infection in the last month were excluded. Whole blood of selected subjects was collected into heparin-treated anticoagulation tubes. Plasma was isolated by centrifuging the whole blood at 1000g for 15 min. Hemolyzed samples were not used. All blood samples used were not subjected to a freeze-thaw cycle at any step in the process. Plasma ATP levels were assessed with an ATP assay bioluminescence detection kit (Beyotime).