StayBrite™ Highly Stable ATP Bioluminescence Assay Kit (BioVision Inc., Milpitas, CA, USA) were used to measure the intracellular level of ATP. After treatment with various concentrations of PD for 24 h, HCC cells were trypsinized and subjected to 100 µL of reaction buffer/104 cells followed by pelleting at max speed for 30 sec to remove debris. Supernatant was placed into 96-well plates (10 µL/well) and 90 µL of reaction mix was then added into each well. The luminescence intensity was measured using SpectraMax M5 (Molecular Devices, San Jose, CA, USA).
Staybrite highly stable atp bioluminescence assay kit
Manufactured by Abcam
Sourced in United States
The StayBright™ Highly Stable ATP Bioluminescence Assay Kit is a laboratory equipment product designed for the detection and quantification of ATP. It utilizes a bioluminescent reaction to generate light that is proportional to the amount of ATP present in the sample. The kit provides the necessary reagents and components to perform the assay.
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8 protocols using staybrite highly stable atp bioluminescence assay kit
1
Quantifying Intracellular ATP Levels in HCC Cells
2
Intracellular ATP Measurement in BMDM
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BMDM were infected and treated as indicated, with IAV infected samples collected at 12 and 24 h post infection. LPS + ATP samples were collected at 4 h post LPS treatment with ATP added for the final 30 min. Samples were analyzed using a StayBrite Highly Stable ATP Bioluminescence Assay kit (BioVision, K791−100) according to the kit instructions. To collect cell samples, the culture supernatant was removed, cells were washed 3x with 5 mL DPBS, and then, 100 μL of 1X RIPA buffer was added to each well. Cells lysate was collected and centrifuged at 12,000xg for 30 s. 10 μL of each cell lysate was pipetted into enzyme/buffer mix, and then analyzed quickly using the GloMax Jr. by ProMega on the GloBrite module, in RLUs.
3
ATP Quantification in MII Oocytes
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MII oocytes were washed in nuclease-free water and transferred individually into a tube in a total volume of 10 µL and stored at −20 °C until ATP testing. The concentration of ATP in the oocytes was determined as previously described [21 (link)], using a specific luminescence kit according to the manufacturer’s protocol (StayBrite™ Highly Stable ATP Bioluminescence Assay Kit, BioVision, K791-100; Milpitas, CA, USA).
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ATP Measurement in Neuronal-Astrocytic Co-Cultures
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ATP Bioluminescence Assay for Retinas
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The amount of ATP was measured using a StayBrite Highly Stable ATP Bioluminescence Assay Kit (BioVision). Each experiment was run in duplicate. ATP per retina was calculated and plotted compared with control retinas. Statistical analysis was performed using Student's t-test.
6
Metabolic Profiling of Mouse Blood and Liver
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Mouse blood samples were collected from the submandibular vein of mice. Blood glucose and lactate levels were determined using the OneTouch Ultra®2 glucose meter (LifeScan) and the Lactate Scout meter (Lactate.com), respectively. Blood concentrations of ketone bodies were measured using the FreeStyle Precision Neo Blood Glucose and Ketone Monitoring System. Blood was then incubated at RT for 30 min before centrifugation at 2348 × g for serum separation. Serum was stored at −80 °C before use. Serum insulin level was measured using an Ultra Sensitive Mouse Insulin ELISA Kit (cat. no. 90080; Crystal Chem). Serum TG and FFA levels were assessed using a Triglyceride Assay Kit (cat. no. ab65336; Abcam) and a Free Fatty Acid Assay Kit (cat. no. ab65341; Abcam), respectively, as per the manufacturer’s recommendations. Serum L-Amino Acid levels were measured using a L-Amino Acid Assay Kit (ab65347). Liver TG and L-Amino Acid levels were determined using a Triglyceride Assay Kit (cat. no. ab65336; Abcam) and a L-Amino Acid Assay Kit (cat. no. ab65347; Abcam), respectively. Hepatic ATP contents were measured using the StayBrite™ Highly Stable ATP Bioluminescence Assay Kit (catalog no. K791-100; BioVision).
7
ATP Release Quantification Assay
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ATP release was determined using a StayBrite Highly Stable ATP Bioluminescence Assay kit (BioVision) to measure the ATP concentration in the culture medium according to the manufacturer's instructions. Cells were plated at 50 000‐60 000 cells/well in 200 μL of standard culture medium in a 96‐well plate and incubated overnight. Next day, cells were subjected to starvation by replacing the culture medium with fresh media without FBS and incubating for 2 hours. Cells were treated with 100 μM suramin for 30 minutes at 37°C in order to prevent ATP hydrolysis, as described in a previous study.38 After cells were exposed to 1 μM Yoda1 for 30 minutes at 37°C, the culture medium were collected, placed in 1.5‐mL Eppendorf tubes on ice, and centrifuged at 11 000g for 5 minutes at 4°C. The supernatants were transferred to a 96‐well plate with 10 μL per well in triplicate for each condition. The luminescence intensity was measured using a Flex‐Station III microplate reader. The ATP concentration was derived using a standard curve constructed using 10, 30, 100, 1000, and 10 000 nM ATP.
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Bioluminescent ATP Detection Protocol
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The Staybrite highly stable ATP bioluminescence assay kit from Biovision was used as directed by the manufacturer.
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