OE33 P and OE33 R cells (pre and 24 h post irradiation with 2 Gy) were seeded in 96-well white-walled plates (15,000 cells/well) and allowed to adhere overnight. Relative intracellular ATP levels were measured using the luminescence-based ATPLite assay system (Perkin Elmer), as per the manufacturer’s instructions. Luminescence was measured using a Wallac Victor2 1420 multilabel counter. An additional plate was set up concurrently and a crystal violet assay was performed to normalize ATP measurements to cell number.
Atplite assay system
Manufactured by PerkinElmer
Sourced in United States, Denmark
The ATPlite assay system is a luminescence-based detection platform designed for the quantitative measurement of adenosine triphosphate (ATP) in a variety of sample types. The system utilizes a bioluminescent reaction to generate light output proportional to the amount of ATP present in the sample.
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5 protocols using atplite assay system
1
Measuring Intracellular ATP in Irradiated Cells
2
Quantification of Platelet ATP Levels
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ATP levels were measured by chemiluminescence using the ATPLite Assay System (PerkinElmer, Boston, MA). ATPLite reagent was added directly to platelet suspensions or supernatants at a 1∶10 dilution and luminescence was read using a SpectraFluor Plus plate reader (Tecan Austria Gmbh, Grodïg, Austria).
3
Measuring Intracellular ATP Levels
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Cells were seeded at a density of 10,000 cells/well in 96-well white-walled plates and allowed to adhere overnight. Relative intracellular ATP levels were measured using the luminescence-based ATPLite™ assay system (Perkin Elmer), as per the manufacturer’s instructions. Luminescence was measured using a Wallac Victor2 1420 multilabel counter. An additional plate was set up concurrently and a crystal violet assay was performed to normalise ATP measurements to cell number.
4
Enzymatic activity and mitochondrial function
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Complex (I, III, IV, and V) enzyme activity in cell extracts was measured by the single‐wavelength spectrophotometric assay using detection kits (cat. no. BC0510, BC3240, BC0945 and BC1445, Beijing Solarbio Science & Technology) according to the manufacturers’ protocols. Briefly, 5 × 106 cells were collected and homogenized in lysis buffer. After two steps of centrifugation (600 × g for 10 min and then 11, 000 × g for 15 min) at 4˚C, the precipitate was collected for ultrasonic crushing and then added into the corresponding substrate to start the enzymatic reaction. The absorbance values at different wavelengths (I: 340 nm; III: 550 nm; IV: 550 nm; V: 660 nm) of the metabolic product was detected using a spectrophotometer (Thermo Fisher Scientific Inc.).
The OCR was measured using a 782 Oxygen Meter (Strathkelvin Instruments, Motherwell, UK) according to the manufacturer's instructions. The amplified signal was recorded at sampling intervals of 0.5 s.
Mitochondrial ATP was measured as previously described [34 (link)]. Briefly, the abundance of ATP was determined using the ATPlite assay system (PerkinElmer Inc., Waltham, MA, USA). Basal luminescence was recorded before the use of luciferin, and then cells were perfused with 20 μmol/L luciferin to record mitochondrial luminescence [35 (link)].
The OCR was measured using a 782 Oxygen Meter (Strathkelvin Instruments, Motherwell, UK) according to the manufacturer's instructions. The amplified signal was recorded at sampling intervals of 0.5 s.
Mitochondrial ATP was measured as previously described [34 (link)]. Briefly, the abundance of ATP was determined using the ATPlite assay system (PerkinElmer Inc., Waltham, MA, USA). Basal luminescence was recorded before the use of luciferin, and then cells were perfused with 20 μmol/L luciferin to record mitochondrial luminescence [35 (link)].
5
Chlorinated Nucleoside Cytotoxicity Assay
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ATP was quantified by luminescence using the ATPlite assay system (Perkin Elmer, Ballerup, Denmark). The INS-1E cells (1.2 × 105/well in a 24-well plate) were exposed to each chlorinated nucleoside (20 µM) in complete RPMI-1640 and incubated for 24 h at 37 °C, 5% CO2. Following exposure, the cells were washed, detached by gentle scraping, and resuspended in HBSS at a final volume of 250 µL. Each sample (100 µL) was transferred to a black 96-well plate, followed by the addition of the supplied cell lysis solution (50 μL) and mixing on an orbital shaker for 5 min. Luminescence was measured using a SpectraMax i3 plate reader (Molecular Devices) after the addition of the substrate solution (50 μL), mixing on the orbital shaker for 5 min, and dark adaption for 10 min.
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