The Fo-F1 ATP synthase (ATP Synthase) activity was evaluated on 20,000 cells permeabilized with 0.03 mg/mL digitonin for 1 min. Cells were incubated for 10 min in a medium containing: 50 mM Tris-HCl pH 7.4, 50 mM KCl, 1 mM EGTA, 2 mM MgCl2, 0.6 mM ouabain, 0.25 mM di(adenosine)-5-penta-phosphate (Ap5A, adenylate kinase inhibitor), and 25 μg/mL ampicillin (0.1 mL final volume). ATP synthesis was induced by the addition of 10 mM pyruvate plus 5 mM malate, and 0.1 mM ADP to stimulate the pathway composed by Complexes I, III, and IV. The reaction was monitored for 2 min, every 30 s, with a luminometer (GloMax® 20/20n Luminometer, Promega Italia), by the luciferin/luciferase chemiluminescent method, with ATP standard solutions between 10−8 and 10−5 M (luciferin/luciferase ATP bioluminescence assay kit CLS II, Roche, Basel, Switzerland). Data were expressed as nmol ATP/min/106 cells [5 (link)].
Luciferin luciferase atp bioluminescence assay kit cls 2
Manufactured by Roche
Sourced in Switzerland
The Luciferin/luciferase ATP bioluminescence assay kit CLS II is a laboratory equipment product designed for the detection and quantification of ATP. It utilizes the bioluminescent reaction between luciferin and luciferase to produce light, which is proportional to the amount of ATP present in the sample. The kit provides the necessary reagents and materials to perform this ATP-based bioluminescence assay.
Automatically generated - may contain errors
Lab products found in correlation
Glomax 20 20n luminometer, by Promega (4 mentions)
Succinate, by Merck Group (2 mentions)
Glomax 20 20 luminometer, by Promega (2 mentions)
Pyruvate, by Merck Group (1 mentions)
Malate, by Merck Group (1 mentions)
Ouabain, by Merck Group (1 mentions)
Di adenosine 5 penta phosphate, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
6 protocols using luciferin luciferase atp bioluminescence assay kit cls 2
1
ATP Synthase Activity Evaluation
2
Evaluating Cellular Oxidative Phosphorylation
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Oxidative phosphorylation (OxPhos) metabolism was evaluated as oxygen consumption rate (OCR) and ATP synthesis through FoF1 ATP-synthase. For each assay, 105 cells, permeabilized with 0.03 mg/mL digitonin for 1 min, were employed. In both cases, 10 mM pyruvate plus 5 mM malate (#P4562 and #M8304, respectively, Sigma-Aldrich, St. Louis, MO, USA) or 20 mM succinate (#S7501, Sigma-Aldrich, St. Louis, MO, USA) was used as a respiratory substrate to stimulate the respiratory pathway led by Complex I or by Complex II, respectively.
OCR was measured with an amperometric electrode (Unisense Microrespiration, Unisense A/S, Aarhus, Denmark) [14 (link),57 (link)].
ATP synthesis was monitored with a luminometer (GloMax ® 20/20 Luminometer, Promega Italia, Milano, Italy) by the luciferin/luciferase chemiluminescent method (luciferin/luciferase ATP bioluminescence assay kit CLS II, Roche, Basilea, Switzerland) for 2 min every 30 s. ATP standard solutions in a concentration range between 10−8 and 10−5 M were used for the calibration curve [14 (link),57 (link)].
OCR was measured with an amperometric electrode (Unisense Microrespiration, Unisense A/S, Aarhus, Denmark) [14 (link),57 (link)].
ATP synthesis was monitored with a luminometer (GloMax ® 20/20 Luminometer, Promega Italia, Milano, Italy) by the luciferin/luciferase chemiluminescent method (luciferin/luciferase ATP bioluminescence assay kit CLS II, Roche, Basilea, Switzerland) for 2 min every 30 s. ATP standard solutions in a concentration range between 10−8 and 10−5 M were used for the calibration curve [14 (link),57 (link)].
3
Evaluation of Mitochondrial ATP Synthesis
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Evaluation of the Fo–F1 ATP synthase activity was performed as previously described. Briefly, 200,000 cells were incubated for 10 min in a medium containing 10 mM Tris–HCl (pH 7.4), 100 mM KCl, 5 mM KH2PO4, 1 mM EGTA, 2.5 mM EDTA, 5 mM MgCl2, 0.6 mM ouabain and 25 mg/ml ampicillin, and 10 mM pyruvate plus 5 mM malate to stimulate the pathway composed by complexes I, III and IV. ATP synthesis was induced by the addition of 0.1 mM ADP. The reaction was monitored every 30 s for 2 min using a luminometer (GloMax® 20/20n Luminometer, Promega Italia, Milan, Italy) for the luciferin/luciferase chemiluminescent method, with ATP standard solutions used at concentrations between 10−8 and 10−5 M (luciferin/luciferase ATP bioluminescence assay kit CLSII, Roche, Basel, Switzerland). Data are expressed as nmol ATP produced/min/106 cells.
4
Evaluating F₀F₁ ATP-synthase Activity
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The FoF1 ATP-synthase activity was evaluated by incubating 105 cells at 37 °C for 10 min. Incubation was done in a medium containing 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 1 mM EGTA, 2 mM MgCl2, 0.6 mM ouabain (#O0200000, Sigma-Aldrich, St. Louis, MO, USA), 0.25 mM di(adenosine)-5-penta-phosphate (an adenylate kinase inhibitor, #D1387, Sigma-Aldrich, St. Louis, MO, USA)), and 25 μg/mL ampicillin (0.1 mL final volume, #A9393, Sigma-Aldrich, St. Louis, MO, USA). Additionally, 10 mM pyruvate and 5 mM malate (#P4562 and #M8304, respectively, Sigma-Aldrich, St. Louis, MO, USA) or 20 mM succinate (#S7501, Sigma-Aldrich, St. Louis, MO, USA) were added to stimulate the two different respiratory pathways. The reaction was observed, using the luciferin/luciferase chemiluminescent method (luciferin/luciferase ATP bioluminescence assay kit CLS II, Roche, Switzerland), with a luminometer (GloMax® 20/20 Luminometer, Promega Italia, Italy), for 2 min every 30 s. For the calibration, ATP standard solutions in a concentration range between 10−8 and 10−5 M were used. Data were expressed as nmol ATP/min/106 cells.
5
Measuring ATP Synthase Activity
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To evaluate the ATP synthase activity, 100,000 cells were incubated for 10 min at 37 °C in a medium containing 10 mM Tris–HCl, pH 7.4, 100 mM KCl, 5 mM KH2PO4, 1 mM EGTA, 2.5 mM EDTA, 5 mM MgCl2, 0.6 mM ouabain, and 25 mg/ml ampicillin. Afterward, ATP synthesis was induced by the addition of 10 mM pyruvate plus 5 mM malate or 20 mM succinate to stimulate the pathways composed by complexes I, III, and IV or complexes II, III, and IV, respectively. The reaction was monitored for 2 min, every 30 s, in a luminometer (GloMax® 20/20n Luminometer, Promega Italia, Milano, Italy), by the luciferin/luciferase chemiluminescent method, with ATP standard solutions between 10–8 and 10–5 M (luciferin/luciferase ATP bioluminescence assay kit CLSII, Roche, Basel, Switzerland). Data were expressed as nmol ATP produced/min/106 cells [35 (link)]. The oxidative phosphorylation efficiency (P/O ratio) was calculated as the ratio between the concentration of the produced ATP and the amount of consumed oxygen in the presence of respiring substrate and ADP. When the oxygen consumption is completed devoted to the energy production, the P/O ratio should be around 2.5 and 1.5 after pyruvate + malate or succinate addition, respectively [36 (link)].
6
Quantifying Mitochondrial ATP Synthesis
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To evaluate the Fo-F1 ATP synthase activity, 200, 000 H9c2 cells were incubated for 10 min at 37°C in a medium containing: 10 mM Tris-HCl pH 7.4, 100 mM KCl, 5 mM KH 2 PO 4 , 1 mM EGTA, 2.5 mM EDTA, 5 mM MgCl 2 , 0.6 mM ouabain, and 25 mg/ml ampicillin. Afterward, ATP synthesis was induced by the addition of 10 mM pyruvate plus 5 mM malate or 20 mM succinate, to stimulate the pathways composed by complexes I, III and IV pathway or complexes II, III and IV, respectively. To start the reaction 0.08 mM ADP was added. The reaction was monitored for two minutes, every 30 sec, in a luminometer (GloMax® 20/20n Luminometer, Promega Italia, Milano, Italy), by the luciferin/luciferase chemiluminescent method, with ATP standard solutions between 10 -8 and 10 -5 M (luciferin/luciferase ATP bioluminescence assay kit CLSII, Roche, Basel, Switzerland). Data were expressed as nmol ATP produced/min/10 6 cells [14] .
The oxidative phosphorylation efficiency (P/O ratio) was calculated as the ratio between the concentration of produced ATP and the amount of consumed oxygen in the presence of respiring substrates and ADP. When oxygen consumption is completed devoted to energy production, the P/O ratio is around 2.5 and 1.5 after pyruvate + malate or succinate addition, respectively [15] .
The oxidative phosphorylation efficiency (P/O ratio) was calculated as the ratio between the concentration of produced ATP and the amount of consumed oxygen in the presence of respiring substrates and ADP. When oxygen consumption is completed devoted to energy production, the P/O ratio is around 2.5 and 1.5 after pyruvate + malate or succinate addition, respectively [15] .
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