Cells were washed and then incubated in Seahorse XF RPMI medium with 1 mM glutamine, 25 mM glucose and 1 mM sodium pyruvate. Cells were then mock treated with PBS or with 2-deoxy-D-glucose and sodium azide at final concentrations of 6 mM and 10 mM for 1 h to block ATP production via glycolysis and oxidative phosphorylation, respectively. Cells were then washed with 1× PBS, harvested in 1× reporter lysis buffer (RLB, Promega), flash frozen in liquid nitrogen, boiled at 99°C for 15 min, centrifuged at 16168 g , and supernatant retained. 25 µl of supernatant was used to quantify protein concentration using a Pierce BCA assay (Thermo Fisher 23227) for normalization. Another 25 µl was taken and further diluted in 500 µl of 1× RLB. 10 μl of this dilution was used to detect cellular ATP levels in 90 µl of reaction buffer using a luciferase-based ATP determination kit (Thermo Fisher Scientific, A22066). Luminescence was read using a Perkin Elmer Victor2 1420 multi-labeled plate counter. All reactions were performed in triplicate and a standard curve was generated using known amounts of ATP to calculate ATP concentrations in the samples.
Luciferase based atp determination kit
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Luciferase-based ATP determination kit is a laboratory tool used for the quantitative detection of adenosine triphosphate (ATP) levels. It employs the luciferase enzyme to catalyze a bioluminescent reaction, which is proportional to the amount of ATP present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax m2, by Molecular Devices (3 mentions)
Victor2 1420, by PerkinElmer (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions)
Spectramax m5 spectrophotometer, by Molecular Devices (1 mentions)
Bradford reagent, by Thermo Fisher Scientific (1 mentions)
Varioskan flash microplate reader, by Thermo Fisher Scientific (1 mentions)
Powerwave microplate spectrophotometer, by Agilent Technologies (1 mentions)
Hsp70 high sensitivity elisa kit, by Abcam (1 mentions)
Synergy ht multi detection microplate reader, by Agilent Technologies (1 mentions)
White bottomed 96 well plate, by Corning (1 mentions)
Synergy ht plate reader, by Agilent Technologies (1 mentions)
8 protocols using luciferase based atp determination kit
1
Quantifying Cellular ATP Levels
2
Galactose-Induced ATP Measurement
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Cells were grown in a 96-well white walled plate in standard media to 80% confluence. Media was replaced with glucose free media supplemented with galactose. Steady state ATP levels were measured using the luciferase based ATP determination kit (Thermo Fisher Scientific). ATP concentration in cell lysates was measured by luminescence on a SpectraMax M5 spectrophotometer (Molecular Devices) and normalized to protein concentration by BCA (Thermo Fisher Scientific).
3
Quantifying Tumor Interstitial Fluid Biomarkers
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The tumor interstitial fluid was obtained by digesting tumors as described above. After removing cells by centrifugation, the supernatant was further purified by centrifugation at maximum speed for 10 min and stored at −70 °C until use. The protein concentration of the tumor effusate was measured using Bradford reagent (Thermo Fisher Scientific). In the subsequent measurements, an equal amount of protein was used. The hsp70 concentration was measured using a hsp70 High-Sensitivity ELISA kit (Abcam). The HMGB1 concentration was determined using a specific ELISA kit from Aviva Systems Biology (San Diego, CA, USA) in accordance with the manufacturer’s protocol. Absorbance was measured at 450 nm with PowerWave microplate spectrophotometer (BioTek, Winooski, VT). ATP content was evaluated using a luciferase-based ATP determination kit (Thermo Fischer Scientific) following the manufacturer’s instructions. Luminescence was measured with a Varioskan Flash microplate reader (Thermo Fischer Scientific).
4
Quantifying Cardiac ATP Levels
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ATP concentration in heart tissue was measured using luciferase‐based ATP determination kit (Invitrogen, Paisley, UK) according to the manufacturer instructions. Luminescence was measured at 560 nm using microplate reader/multidetection reader (SpectraMax M2; Molecular Devices, Wokingham, UK).
5
Quantifying Total ATP Content in Cells
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Total ATP content was measured using luciferase-based ATP determination kit according to manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.). For ATP measurement, 1×106 cells were harvested, centrifuged at 100 × g for 5 min at room temperature and re-suspended in 100 µl buffer (25 mM Tris; pH 7.4; 150 mM EDTA). The re-suspended cells were boiled at 100°C for 5 min, followed by centrifugation at 10,000 × g for 5 min at room temperature. The supernatant was collected, added to the luciferin-luciferase mixture and luminescence was measured in BioTek Synergy HT Multi-detection Microplate reader. The ATP concentration was determined using a standard ATP/luminescence curve ranging from 0.001–10 mM ATP, normalized to total protein and calculated as relative fold change between CRC cells.
6
Quantifying ATP Release After Injury
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To determine concentration of ATP released after injury, HCLE cells were plated on culture-treated plastic and grown to confluence in KSFM containing growth supplements. The growth supplements were removed from the media 24 hours before wounding. To wound the cells, a comb made from plastic gel-loading tips was used to make a scratch wound, and the media was collected every 20 minutes and clarified by centrifugation at 663 x g. The clarified media was collected and stored on ice until ready for analysis with a luciferase-based ATP Determination Kit (Invitrogen, Carlsbad, CA). Samples were vortexed and 5 μL aliquots were plated on a white-bottomed 96-well plate (Corning). A reaction buffer (0.5 mM D-luciferin, 1.25 μg/mL firefly luciferase, 25 mM Trycine buffer, pH 7.8, 5 mM MgSO4, 100 μM EDTA, and 1 mM DTT) was prepared immediately before analysis and protected from light. To determine ATP levels, luciferase-generated luminescence was detected using a BioTek Synergy HT plate reader with injector (BioTek, Winooski, VT). A standard curve of ATP was made in KSFM. To ensure equal time for each reaction, 95 μL of reaction buffer was injected into a well and allowed to incubate for four seconds before luminescence was read. ATP levels were calculated from raw luminescence values using the standard curve.
7
Quantifying ATP in Heart Tissue
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ATP concentration in heart tissue was measured using luciferase-based ATP determination kit (Invitrogen, Paisley, UK) according to the manufacturer's instructions. Luminescence was measured at 560 nm using microplate reader/multidetection reader (SPECTRAMAX M2, Molecular Devices, Wokingham, UK).
8
Quantification of ATP in Heart Tissue
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ATP concentration in heart tissue lysates was measured using luciferase-based ATP determination kit (Invitrogen, Paisley, UK) according to the manufacturer's instructions. Luminescence was measured at 560 nm using microplate reader/multidetection reader (SPECTRAMAX M2, Molecular Devices, Wokingham, UK).
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