The extracellular amount of ATP was measured using the ATP determination kit according to the protocol provided by its supplier Invitrogen (ATP determination kit, A22066). Using the conditioned medium of the different treated cell lines and applying the kit that includes D-Luciferin and recombinant firefly luciferase, the luminescence produced by luciferin upon binding with ATP present in the conditioned medium was measured using a Tecan Infinite® M200 PRO plate reader spectrometer (Männedorf, Switzerland) and the i-control™ software.
Atp determination kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Canada, Spain, France
The ATP Determination Kit is a quantitative colorimetric assay designed to measure the concentration of adenosine triphosphate (ATP) in a sample. The kit utilizes the enzyme firefly luciferase to catalyze the reaction between ATP and luciferin, producing light that can be detected and quantified.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions)
Infinite m200 pro, by Tecan (7 mentions)
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Lactate colorimetric fluorometric assay kit, by Abcam (5 mentions)
Glomax 20 20 luminometer, by Promega (5 mentions)
Xf96 extracellular flux analyzer, by Agilent Technologies (5 mentions)
Arl67156, by Merck Group (5 mentions)
Passive lysis buffer (plb), by Promega (5 mentions)
Dc protein assay, by Bio-Rad (4 mentions)
Synergy h1, by Agilent Technologies (4 mentions)
Antimycin a, by Merck Group (4 mentions)
Lactate assay kit, by Merck Group (4 mentions)
Glomax, by Promega (4 mentions)
Glucose assay kit, by Merck Group (3 mentions)
Fluostar optima plate reader, by BMG Labtech (3 mentions)
Infinite m1000 pro, by Tecan (3 mentions)
Potassium chloride (kcl), by Merck Group (3 mentions)
Microplate reader, by Agilent Technologies (3 mentions)
Oligomycin, by Merck Group (3 mentions)
610 protocols using atp determination kit
1
Extracellular ATP Quantification Protocol
2
Extracellular ATP Quantification in Mesangial Cells
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The extracellular amount of ATP was measured using the ATP determination kit according to the protocol provided by its supplier Invitrogen (ATP determination kit, A22066). Using the conditioned medium of mesangial cells and applying the kit that includes D-Luciferin and recombinant firefly luciferase, the luminescence produced by luciferin upon binding with ATP was measured using a Tecan Infinite® M200 PRO plate reader spectrometer (Männedorf, Switzerland) and i-control™ software.
3
Quantifying Intracellular and Extracellular ATP Levels
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The ATP level was determined with a bioluminescence assay using an ATP Determination Kit (A22066, Invitrogen). Briefly, C. albicans cells were diluted to a final concentration of 3 × 108 CFU/mL with 12.5 mM sodium acetate, incubated with or without different concentrations of LfcinB15 at 37 °C for 1 h, and collected by centrifugation. The supernatant was kept on ice before use for measuring the extracellular ATP level, while the cell pellets were broken, as previously described [30 (link)], for measuring intracellular ATP.
For ATP measurement, the ATP Determination Kit was used according to the manufacturer’s instructions (Invitrogen). Briefly, 1× reaction buffer containing 0.5 mM D-luciferin, 1.25 μg/mL firefly luciferase, and 1 mM DTT was freshly prepared before each experiment, and 10 µL of the supernatant or cell lysate was mixed with 90 µL of the reaction buffer. Then, the mixture was placed into each well of a black 96-well microplate, and luminescence was read using a Victor3™ Plate Reader (PerkinElmer) at 560 nm. A standard curve of increasing ATP concentrations (0, 0.01, 0.1, 1 μM) was created. Signals represented at least three independent experiments were obtained and the ATP concentration of each sample was calculated from the standard curve.
For ATP measurement, the ATP Determination Kit was used according to the manufacturer’s instructions (Invitrogen). Briefly, 1× reaction buffer containing 0.5 mM D-luciferin, 1.25 μg/mL firefly luciferase, and 1 mM DTT was freshly prepared before each experiment, and 10 µL of the supernatant or cell lysate was mixed with 90 µL of the reaction buffer. Then, the mixture was placed into each well of a black 96-well microplate, and luminescence was read using a Victor3™ Plate Reader (PerkinElmer) at 560 nm. A standard curve of increasing ATP concentrations (0, 0.01, 0.1, 1 μM) was created. Signals represented at least three independent experiments were obtained and the ATP concentration of each sample was calculated from the standard curve.
4
Quantifying ATP and mtDNA Levels
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ATP was measured according to manufactures protocol using the ATP Determination Kit (Molecular Probes). COS-7 cells were transfected with siRNAs as previously described and homogenized in ATP assay buffer at 48 hours post transfection, or 5 whole zebrafish embryos were homogenized in ATP assay buffer at 24 hours post fertilization. Homogenates were spun at 12,000 g for 5 min to pellet tissues debris. 10 ul supernatant were added to a 97 well dish containing 90 ul of the luciferase reaction mix from the ATP Determination Kit (Molecular Probes). Luminescence was immediately read on a luminometer. ATP values were calculated by comparison to a standard curve generated from a series of ATP concentrations. Values were then normalized by the total amount of protein present in each sample. Samples were run in triplicate. mtDNA levels were quantified by quantitative PCR as described38 (link).
5
Measuring Biofilm Cell Lysis by ATP
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To measure cell lysis, we used the luminescence assay described in reference 29 (link) to measure increases in extracellular ATP levels using a commercial ATP determination kit (Molecular Probes). Prior to measuring cell lysis, biofilms were grown for 24 h with different concentrations of antibiotics, as described previously, in a 96-well plate. We then washed the plate twice with nuclease-free water and removed excess liquid. After washing, 10 μl of nuclease-free water was added to each well and biofilms were scraped by using inoculation loops or pipette tips. Solutions were transferred to a new 96-well white polystyrene plate (Nunc F96 MicroWell; Thermo Scientific), and 90 μl of ATP standard assay solution from the ATP determination kit (Molecular Probes) was added to each sample. Luminescence was measured with a plate reader.
6
In Vitro and Cell-Based NDPK Assays
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In vitro NDPK assay; 5 ng of recombinant Nm23-H1 were pre-incubated with 5 µM GTP (or UTP) and NMac1 in NDPK assay buffer (20 mM HEPES, 3 mM MgCl2) at R.T. for 10 min followed by enzyme reaction by adding 5 µM for 1 min. Enzyme reaction was stopped by 95 °C boiling for 10 min. ATP generations were assessed by ATP determination kit (Molecular probe, USA). Cell based NDPK assay was performed. Cell lysates obtained from 5 × 106 MDA-MB-231 cells which were lysed with NDPK assay buffer with protease inhibitor cocktail, and centrifuged 8,000 rpm at 4 °C for 10 min. 40 µL lysate incubated with NMac1 for 5 min followed by NDPK reaction by adding 50 µM UDP. ATP consumption was assessed by ATP determination kit (Molecular probe, USA). Origin 6.0 (USA) was used for curve fitting of NDPK assay data with logistic dose responsive function.
7
Quantifying ATP and mtDNA Levels
8
Extracellular ATP Quantification in Activated T Cells
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Purified total T cells were activated with anti-CD3/CD28 Dynabeads for 4 days. eATP in the supernatant was quantified using the ATP determination kit (Thermo Fisher Scientific). Briefly, supernatants were incubated with the premade mix including luciferase and its substrate luciferin for 10 minutes at room temperature. Luminescence was read using BioTek Synergy H1. ATP concentrations were calculated from a standard curve.
Purified T cells from the donors with AG or GG genotype at rs10748643 were stimulated in the presence or absence of AS1517499 (100 nM) or IL-4 (20 ng/mL). On day 4, cells were resuspended in medium supplemented with 20 μM ATP. ATP concentrations in the supernatant were determined after incubation for 30 minutes and 1 hour.
Purified T cells from the donors with AG or GG genotype at rs10748643 were stimulated in the presence or absence of AS1517499 (100 nM) or IL-4 (20 ng/mL). On day 4, cells were resuspended in medium supplemented with 20 μM ATP. ATP concentrations in the supernatant were determined after incubation for 30 minutes and 1 hour.
9
Mitochondrial ATP Measurement by Luminescence
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The level of mitochondrial ATP was measured using the luminescence assay ATP Determination Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. The assay was based on luciferase’s absolute requirement for ATP in producing light. Each sample was measured in duplicate, using a microplate reader TECAN Infinite M1000PRO (560 nm). The ATP levels were calculated as luminescence produced in the mitochondria suspension per milligram of mitochondrial protein and were expressed as arbitrary units (AU).
10
Measuring Islet ATP under Glucolipotoxicity
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After being cultured under control or glucolipotoxic conditions in the presence or absence of oltipraz for 48 h, islets were silenced in bath solution containing 6 mmol/L glucose for 1 h. Subsequently, 20 islets per batch were incubated in bath solution with either 0.5 or 15 mmol/L glucose at 37°C for 30 min. Islets were lysed and incubated at 60°C to inactivate enzymes for 20 min. ATP content was measured using a luciferin/luciferase-based assay according to the manufacturer's protocol (ATP determination kit, Invitrogen™, Thermo Fisher Scientific).
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