ATP contents were assessed using a bioluminescence assay kit (Beyotime), according to the manufacturer’s instructions.
Bioluminescence assay kit
Manufactured by Beyotime
Sourced in China
The Bioluminescence Assay Kit is a laboratory tool designed to detect and quantify bioluminescent signals. It provides the necessary reagents and protocols to perform bioluminescence-based experiments, a versatile technique used in various fields of research.
Automatically generated - may contain errors
Lab products found in correlation
Synergy mx, by Agilent Technologies (1 mentions)
Protein carbonyl colorimetric assay kit, by Cayman Chemical (1 mentions)
Cellquest pro software, by BD (1 mentions)
Lipid peroxidation mda assay kit, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
7 protocols using bioluminescence assay kit
1
Quantitative ATP Bioluminescence Assay
2
ATP Quantification in Podocytes
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Contents of ATP were assessed with a bioluminescence assay kit (Beyotime, Shanghai, China). The measurement was conducted as previously described 31 (link). After the treatment, supernatants were removed and podocytes were lysed in the ATP assay buffer for 5 min. Cells from each well were then collected for centrifugation. The supernatant fraction was put into a 96-well clear bottom, black-walled microplate (Corning Incorporated, NY, USA) and then luciferase reagent was added. The luminescence was detected by a luminometer (Synergy, BioTek Instruments, USA).
3
Quantifying Kidney ATP Levels
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ATP was measured using a bioluminescence assay kit (Beyotime Biotechnology, Shanghai, China). Briefly, the fresh kidney samples were lysed in the lysis buffer provided with the kit. The supernatant was collected by centrifugation at 12,000 rpm for 5 min at 4 °C. The concentration of ATP present in the samples was determined by mixing 20 μL of the supernatant with 100 μL of luciferase reagent; the luciferase present in the reagent catalyzes the production of luminescence from ATP and luciferin. The luminescence of each sample was measured on a microplate luminometer (Synergy Mx, BioTek Instruments Inc., Winooski, VT, USA). A standard curve of ATP was prepared using a series of standards of known concentrations; the measured ATP is presented as nmol/mg of protein.
4
Gastric ATP Quantification by Bioluminescence
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The ATP concentration in the gastric sections was measured using a bioluminescence assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's protocol. In brief, gastric tissues were lysed in the appropriate lysis buffer. The supernatant was collected by centrifugation at 12,000 × rpm for 5 min at 4 °C. The ATP concentration in the tissues was examined by mixing 20 μl of the supernatant with 100 μl of luciferase reagent (luciferase can catalyze the generation of luminescence from ATP and luciferin) provided with the assay kit. The luminescence of each sample was measured on a microplate luminometer, and the measured ATP concentration is presented as nmol/mg of protein based on a standard curve of ATP.
5
Bioluminescent ATP Quantification
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ATP was measured using a bioluminescence assay kit (Beyotime Biotechnology, Shanghai, China). After different treatments, the cells were lysed with 200 μl of lysis buffer provided with the kit. The supernatant was collected by centrifugation at 12,000g for 5 min at 4°C. The concentration of ATP was determined by mixing 100 μl of luciferase reagent and 20 μl of supernatant to catalyze the production of luminescence from ATP and luciferin. The luminescence of each sample was measured using a microreader (Tecan Group Ltd., Switzerland).
6
Antioxidant Response of A. flavus to PAW Treatment
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After treatment with the instant-prepared PAW, A. flavus was inoculated onto PDA and cultivated for 2, 3, and 5 d. The SOD activity, CAT activity, and ATP content of A. flavus at different culture times were measured. Mycelia were freeze-dried in liquid nitrogen and grounded. After that, the samples were homogenized in PBS and centrifuged in 7000× g for 15 min at 4 °C. The supernatant was used for the detection of ATP content, total protein concentration, and enzyme activity. The ATP level was measured with a bioluminescence assay kit (Beyotime, Shanghai, China). The total protein concentration of A. flavus was determined by Bradford assay (Protein standard: bovine serum albumin). SOD and CAT activities were detected by commercial kits (Cu/Zn-SOD and Mn-SOD Assay Kit, Catalase Assay Kit, Beyotime, Shanghai, China). The SOD or CAT activities were expressed as the SOD activity or CAT activity divided by total protein concentration.
7
Evaluating Mitochondrial and Oxidative Stress
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MMP loss was evaluated by Mitochondrial membrane potential assay kit with Jc-1 (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. The cells were then assessed using a flow cytometer. The results were analyzed using cellQuest Pro software (version 5.2.1; Bd Biosciences).
ATP level measurement. Intercellular ATP levels in HacaT cells were measured via bioluminescence assay kit (Beyotime, Jiangsu, china) based on provided protocols. Levels of ATP were determined based on luciferase luminescence, after normalizing to total protein content.
Measurement of oxidation biomarkers. Malondialdehyde (MdA) content was assessed using Lipid Peroxidation MdA Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. The amount of 8-oxo-2'-deoxyguanosine (8-oxo-dG), a dNA damage marker, was determined with a Human 8-OhdG ELISA Kit (AMEKO, Shanghai, china), according to the manufacturer's instructions. Protein carbonyl levels were measured by Protein carbonyl colorimetric Assay Kit (cayman chemical, Ann Abor, MI, USA), according to the manufacturer's protocol.
ATP level measurement. Intercellular ATP levels in HacaT cells were measured via bioluminescence assay kit (Beyotime, Jiangsu, china) based on provided protocols. Levels of ATP were determined based on luciferase luminescence, after normalizing to total protein content.
Measurement of oxidation biomarkers. Malondialdehyde (MdA) content was assessed using Lipid Peroxidation MdA Assay kit (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. The amount of 8-oxo-2'-deoxyguanosine (8-oxo-dG), a dNA damage marker, was determined with a Human 8-OhdG ELISA Kit (AMEKO, Shanghai, china), according to the manufacturer's instructions. Protein carbonyl levels were measured by Protein carbonyl colorimetric Assay Kit (cayman chemical, Ann Abor, MI, USA), according to the manufacturer's protocol.
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