ATP concentration was determined using a kit of ENLITEN rLuciferase/Luciferin reagent (FF2021, Promega, Madison, WI, USA) as described (Lu et al., 2016 (link)). Briefly, 30 μg of sample proteins from total protein fractions were suspended in 100 μL of reconstituted rL/L reagent buffer containing luciferase, D-luciferin, Tris-acetate buffer (pH 7.75), EDTA, magnesium acetate, bovine serum albumin, and dithiothreitol. Light emission at 10-second intervals from the reaction was measured in a standard microplate luminometer (PE Applied Biosystems). Relative light units from “background blank” containing rL/L reagent and the homogenization buffer used to prepare the samples were subtracted from the sample light output in the assay. Values of ATP activity levels were determined using an ATP standard curve. CCO activity in the mitochondrial fractions was assessed using an activity assay kit (ab109911, Abcam Inc) according to the instruction of the manufacturer. CCO enzyme was immunocaptured within the 96-well microplate, and activity was determined colorimetrically by measuring the oxidation of reduced cytochrome c at 550 nm absorbance (Bio-Rad Benchmark Plus, Microplate Spectrophotometer). The data of ATP levels and relative CCO activity were quantified as percentage changes versus control group for graphical depiction.
Enliten rluciferase luciferin reagent
Manufactured by Promega
Sourced in United States, Italy
The ENLITEN rLuciferase/Luciferin reagent is a lab equipment product that provides a bioluminescent reporter system. It contains recombinant luciferase enzyme and its substrate luciferin, enabling the detection and quantification of ATP levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Standard microplate luminometer, by Thermo Fisher Scientific (4 mentions)
Wallac victor3 1420 luminometer, by PerkinElmer (3 mentions)
Benchmark plus microplate spectrophotometer, by Bio-Rad (2 mentions)
D luciferin, by Promega (2 mentions)
Fluostar optima microplate luminometer, by BMG Labtech (2 mentions)
Ab109911, by Abcam (1 mentions)
Glomax 20 20, by Promega (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Evans blue, by Merck Group (1 mentions)
Lmax luminometer, by Molecular Devices (1 mentions)
A740003, by Bio-Techne (1 mentions)
Ivis lumina luminometer, by PerkinElmer (1 mentions)
Spectramax luminometer, by Molecular Devices (1 mentions)
Bovine cii, by Chondrex (1 mentions)
Exogenous atp, by Merck Group (1 mentions)
Synergy htx multi mode reader, by Agilent Technologies (1 mentions)
19 protocols using enliten rluciferase luciferin reagent
1
Quantification of ATP and CCO Activity
2
AML Cell Viability Assay
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HL-60, KG-1 or primary AML cells were seeded in 96-well flat bottom plates (1 × 106/mL) and treated with chemotherapeutic agents DNR, Ara-C, Flu and Eto (as described above), for 4 h. Cells were then washed and after 20 h ATP quantification in the supernatants was performed in triplicate using ENLITEN rLuciferase/Luciferin Reagent (Promega, Madison, WI, USA), according to the manufacturer’s instructions. Luminescence was measured at the single-tube luminometer Glomax 20/20 (Promega), with 10-second RLU (relative light units) signal integration time.
3
UVA-Induced Extracellular ATP Release
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Extracellular ATP concentration was measured by using ENLITEN rLuciferase/Luciferin Reagent (Promega). After irradiation, culture supernatant was collected at the indicated time points. Each sample was centrifuged at 600 g for 1 min and 10 μL of the supernatant was used for ATP determination. Luciferin-luciferase reagent (100 μL) was added to the supernatant, and the chemiluminescence was measured with a SpectraMax M5 (Molecular Devices, Orleans, CA). The ATP concentration in each sample was determined by comparing the luminescence of samples with those of standards in the range of 10–6–10–9 M. For the study on inhibitory effects of various inhibitors on the UVA-induced ATP release, cells were pretreated with GdCl3 (50 μM), glibenclamide (100 μM), bafilomycin A (50 nM), CBX (20 μM), AZ11645373 (10 μM), FFA (50 μM), LaCl3 (200 μM), probenecid (500 μM), or trovafloxacin (30 μM) for 30 min. At 1 min after irradiation, the supernatants were collected and the ATP content was measured according to the procedures as mentioned above.
4
Extracellular ATP Measurement Protocol
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Extracellular ATP concentration was measured by using ENLITEN® rLuciferase/Luciferin Reagent (Promega, Madison, WI). After irradiation, culture supernatant was collected at the indicated time points. Each sample was centrifuged at 600 × g for 1 min and 10 μL of the supernatant was used for ATP determination. Luciferin–luciferase reagent (100 μL) was added to the supernatant, and the chemiluminescence was measured with a SpectraMax M5. The ATP concentration in each sample was determined by comparing the luminescence of samples with those of standards in the range of 10−6–10−9 M.
5
Quantifying ATP Levels Utilizing Luciferin-Luciferase
6
Extracellular ATP Measurement in B16F10 Cells
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ATP was measured with the ENLITEN rLuciferase/Luciferin reagent (cat# FF2021, Promega, Milan, Italy) with a Perkin Elmer Wallac Victor3 1420 luminometer (Perkin Elmer, Wellesley, Massachusetts, USA). Briefly, to measure eATP, 5 × 103 B16F10 cells/well were plated in ninety-six-well microplates (cat# 655077, Greiner Bio-One). Following adhesion, cells were incubated for 48 h in the presence or absence of serum, or alternatively treated with 1 mM HC in serum-containing medium. Then, supernatants were removed, and 50 μL of diluent buffer (FireZyme, San Diego, CA, USA) to stabilize ATP, and 50 μL of ENLITEN reagent were added to each well. To measure total iATP content, cells were lysed with 5 μL of milli-Q water and supplemented with 45 μL of diluent buffer (FireZyme) and 50 μL of ENLITEN reagent. Extracellular ATP was also measured with the pmeLuc probe in the IVIS Lumina luminometer. 50 × 103 B16F10-pmeLuc cells/well were plated in 24-well plate and cultured in the presence or absence of serum, or treated with HC (1 mM) in serum-containing medium. D-Luciferin (Promega) was added to each well at a concentration of 300 μg/mL. Luminescence is expressed as total photons measured during a 5 min acquisition.
7
Quantitative ATP Measurement in FBS
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ATP was measured with the ENLITEN rLuciferase/Luciferin reagent (cat # FF2021, Promega, Milan, Italy) with a PerkinElmer Wallac Victor3 1420 luminometer (PerkinElmer, Wellesley, MA, USA). Briefly, 100 µL of FBS (or BSA solution) was placed in 96-well microplates (cat # 655077, Greiner Bio-One Italia, Milan, Italy) and then 2 μL of lysis buffer (not in the case of BSA) and 100 μL of ENLITEN reagent were added to each well. In control experiments, 100 µL of FBS was treated with 1 µg of apyrase for 10 min, before being used for ATP measurement.
8
Measuring Extracellular ATP Release
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Adenosine triphosphate release was measured using the ENLITEN® rLuciferase/Luciferin reagent (Promega). Granule cells were maintained for 30 min at 37°C in Mg2+-free Locke’s buffer containing 100 μM ARL67156, a competitive inhibitor of ecto-ATPases (Levesque et al., 2007 (link)), and subsequently, 50 μl of extracellular medium was collected to measure the basal ATP levels. The cells were then stimulated with 10 μM ionomycin and after 5 min, 50 μl of the extracellular medium was collected to measure ATP concentration after stimulation. The samples were centrifuged at 600 × g for 5 min at 4°C and 10 μl of supernatant were transferred onto 96-well plate placed on ice. When indicated, cells were incubated with 2 μM Evans Blue (Sigma–Aldrich) for 1 h (Loiola and Ventura, 2011 (link)). The plate was then situated in a FLUOstar OPTIMA Microplate Luminometer (BMG LABTECH GmbH) and 100 μl of rLuciferase/Luciferin reagent was automatically injected into each well at RT. The ATP concentration was estimated by interpolation from a linear standard curve.
9
Extracellular ATP Quantification Protocol
10
Measuring ATP Levels in Cell Cultures
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ATP was measured in the culture supernatants with ENLITEN rLuciferase/Luciferin reagent (Promega), as per the manufacturer’s instructions. Briefly, 50 × 103 B16 cells and peritoneal macrophages per well were plated in six-well plates. Following adhesion cells were incubated for 48 h in the presence of vehicle or A740003 (Tocris Bioscience) 20 µM, corresponding to in vivo administered concentration. Luminescence from cell supernatants was measured following addition of 100 μl of ENLITEN reagent per well, in a Perkin Elmer Wallac Victor3 1420 luminometer (Perkin Elmer, Wellesley, Massachusetts, USA). Peri-cellular ATP levels were also measured, thanks to the pmeLUC probe and the use of an IVIS Lumina luminometer (Caliper). d -Luciferin (Promega) was added to all wells at a concentration of 60 μg/ml. Luminescence data were expressed as total photons measured in a 5 min acquisition.
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