Both the ADP/ATP ratio and the total cellular ATP were estimated using the bioluminescence-based ADP/ATP assay kit (Sigma MAK135) following the manufacturer's protocol. In brief, 1 × 106 cells per sample were harvested and washed once with PBS-G (1× PBS plus 6-mM glucose). Cells were resuspended in 10 μl of 1× PBS-G and transferred into a white flat-bottom 96-well microtiter plate. Luminescence was recorded in an Orion II microplate luminometer (Titertek-Berthold). The ATP levels and calculated ADP/ATP ratios of RNAi-induced/tetracycline-depleted cells were normalized to those of control cells and expressed in percentage. The values were plotted and analyzed statistically using GraphPad Prism 8.0 software.
Adp atp assay kit
Manufactured by Merck Group
The ADP/ATP assay kit is a laboratory tool used to measure the levels of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) in biological samples. It provides a quantitative assessment of the energy status within cells or tissues.
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Lab products found in correlation
Orion 2, by Berthold Technologies (1 mentions)
Prism 8, by GraphPad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Rabbit anti p 4e bp1 t37 46, by Cell Signaling Technology (1 mentions)
Aicar, by Merck Group (1 mentions)
Angiotensin 2, by Merck Group (1 mentions)
Rabbit anti ampkα, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Rabbit anti p62, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p ampkα, by Cell Signaling Technology (1 mentions)
Rabbit anti stat3, by Cell Signaling Technology (1 mentions)
Rabbit anti p mtor, by Cell Signaling Technology (1 mentions)
Rabbit anti pstat3, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rabbit anti β actin, by Santa Cruz Biotechnology (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Atp assay kit, by Beyotime (1 mentions)
Glomax luminometer, by Promega (1 mentions)
Ow cytometry analysis, by Beckman Coulter (1 mentions)
6 protocols using adp atp assay kit
1
Quantifying ADP/ATP Ratio and ATP Levels
2
Intracellular Signaling Pathway Analysis
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Media and reagents were acquired from the following sources: DMEM and Trypsin from Corning; FBS from Gibco; cell lysis buffer from Cell Signaling Technology; angiotensin II, WP1066, AICAR and compound C from EMD Millipore; (GFP)-fused LC3B (pEGFP-LC3) from Addgene; and ADP/ATP assay kit from Sigma. Antibodies were acquired from the following sources: rabbit anti-p-STAT3 (Tyr705), rabbit anti-p-STAT3 (Ser727), rabbit anti-STAT3, rabbit anti-p-mTOR (Ser2448), anti-mTOR, rabbit anti-p-AMPKα (T172), rabbit anti-AMPKα, rabbit anti-p-JAK2 (Tyr1007/1008) and rabbit anti-p-4E-BP1 (T37/46) from Cell Signaling; rabbit anti-LC3Bfrom Novus Biologicals; rabbit anti-β-Actin and rabbit anti-p62 from Santa Cruz Biotechnology.
3
ADP/ATP Ratio Assay in BV-2 Cells
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An ADP/ATP assay kit (Sigma-Aldrich) was used in the ADP/ATP ratio assay. In brief, BV-2 cells were seeded in a 12-well plate (1×105 cells/well). After LPS stimulation for 16 h, the cells were digested and collected in a centrifuge tube. Then, the cells were seeded in a 96-well plate with a white background (1×104 cells/well). The ATP reagent was prepared and added to each well. The samples were placed at room temperature for 1 min and measured by a luminescence reporter assay system (Promega, Madison, WI, USA). The value was recorded as [(RLU)A]. After 10 min, the samples were measured again as [(RLU)B]. After recording [(RLU)B], 5 mL of ADP reagent was prepared and added to samples without delay. Then, the samples were incubated at room temperature for 1 min before the luminescence was detected as [(RLU)C]. The ADP/ATP ratio was calculated following the manufacturer’s instructions.
4
Measuring ATP and ADP/ATP Levels
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ATP levels were measured using ATP assay kit (Beyotime). The ADP/ATP ratio was analysed using the ADP/ATP assay kit (Sigma).
5
Measuring ADP/ATP Ratio in Human Cells
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For the measurement of the ADP/ATP ratio in human cells, following the collection of cells in PBS as described, the ADP/ATP ratio was determined using the ADP/ATP Assay Kit (Sigma-Aldrich, MAK135-1KT). The assay was conducted on a Glomax Luminometer (Promega), following the guidelines provided by the manufacturer.
6
BV2 Cell-Based ADP/ATP and Viability Assays
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An ADP/ATP Assay Kit (Sigma-Aldrich) was used in the ADP/ATP ratio assay. In brief, BV2 cells were seeded in a 12-well plate (1 × 10 5 cells/well). After LPS or/and compound stimulation for 16 hours, the cells were digested and collected in a centrifuge tube. Then, the cells were seeded in a 96-well plate with a white background (1 × 10 Coculture BV2 cells were cultured in a 6-well plate at a density of 2.0 × 10 5 cells/well. Twenty-four hours later, the supernatant was collected as conditioned medium prior to LPS stimulation, and the compounds were stimulated for 6 hours. MES23.5 dopaminergic neural cells were cultured in a 96-well plate (2.0 × 10 4 cells/well), and after 12 hours, the conditioned medium was added to MES23.5 for 24 hours. Then, cell viability was measured by MTT assay and ow cytometry.
Flow cytometry BV2 cells were treated with 2-DG for 30 min prior to lipopolysaccharide (LPS)-Alexa Fluor® 488 (L23351, Invitrogen, CA, USA) stimulation. After 2 hours, Cells were resuspended and washed in PBS for 3 times.
The cell-associated uorescence was measured by ow cytometry analysis (Beckman Coulter, Brea, CA, USA).
Flow cytometry BV2 cells were treated with 2-DG for 30 min prior to lipopolysaccharide (LPS)-Alexa Fluor® 488 (L23351, Invitrogen, CA, USA) stimulation. After 2 hours, Cells were resuspended and washed in PBS for 3 times.
The cell-associated uorescence was measured by ow cytometry analysis (Beckman Coulter, Brea, CA, USA).
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