ATP content in 3T3-L1 adipocytes or isolated mitochondria was measured using a luciferase-based luminescence enhanced ATP assay kit (Beyotime, Shanghai, China). Cells were washed twice with ice-cold PBS, harvested in 100 μL ice-cold ATP releasing buffer, and centrifuged at 12,000× g for 5 min at 4 °C. Isolated mitochondria (1 mg/mL of protein concentration) were incubated in 0.5 mL of the respiration buffer with 2.5 mM succinate, 2.5 mM malic acid, and 2.5 mM ADP for 10 min. ATP content in cell lysates and the mitochondrial suspension was then determined using a SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices, Sacramento, CA, USA).
Luciferase based luminescence enhanced atp assay kit
Manufactured by Beyotime
Sourced in China
The Luciferase-based luminescence enhanced ATP assay kit is a laboratory equipment product designed to quantify the concentration of ATP (Adenosine Triphosphate) in a sample. The kit utilizes the luciferase enzyme, which catalyzes a reaction that produces luminescence proportional to the amount of ATP present. This assay enables the sensitive detection and measurement of ATP levels in various biological samples.
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4 protocols using luciferase based luminescence enhanced atp assay kit
1
Quantifying Cellular and Mitochondrial ATP
2
Luciferase-based ATP Quantification
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The ATP level was examined using a luciferase-based luminescence enhanced ATP assay kit (Beyotime Institute of Biotechnology, China) following the manufacturer’s instruction. The hepatocytes were washed with ice-cold PBS, lysed with 200 μL lysis buffer, and then centrifuged at 12,000× g for 5 min at 4 °C. ATP content in cell lysates was then determined using a luminescence plate reader.
3
Cellular ATP Quantification Assay
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Cellular ATP content was analyzed with a luciferase-based luminescence-enhanced ATP assay kit (Beyotime, Shanghai, China). Following rotenone treatment for 24 h, cells were rinsed using chilled PBS and lysed with chilled ATP-releasing buffer. Following lysate centrifugation (5 min, 12,000× g, 4 °C), supernatants were collected and combined with an ATP testing working solution. ATP levels in these samples were then measured with a plate reader as above (Molecular Devices, Sacramento, CA, USA).
4
Quantifying ATP in SH-SY5Y Cells
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ATP content in SH-SY5Y cells was measured using a luciferase-based luminescence enhanced ATP assay kit (Beyotime, Shanghai, China). Cells were washed with ice-cold PBS before they were lysed with 100 µL ice-cold ATP releasing buffer. The cell lysates obtained were centrifuged at 12,000× g for 5 min at 4 °C. The supernatants were incubated with the ATP testing working solution provided by the kit. ATP content in cell lysates was determined using a SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices, Sacramento, CA, USA).
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