产品概述 Luminescent ATP Detection Assay Kit (ab113849) is used to measure the level of ATP within the cell. The luminescent ATP assay protocol involves lysis of the cell sample, addition ofluciferase enzyme and luciferin, and measurement of theemitted light using a tube or microplate-based luminometer.This kit irreversibly inactivates ATP degrading enzymes (ATPases) during the lysis step, ensuring that the luminescent signal obtained truly corresponds to the endogenous levels of ATP.Luminescent ATP assay protocol summary:- add ATP standard into standard wells and media into control wells in same plate containing cells to be analyzed- add detergent solution and incubate for 5 min to lyse cells and stabilize ATP- add substrate solution and incubate for 5 min- store plate in dark for 10 min- analyze on luminescence plate readerSpecial Handling Instructions for the ATP Detection Assay KitATP can be found in cells and microbiota on many surfaces. To prevent unintended background, it is recommended to clean bench surfaces and all pipettes to be used during the experiment with 10% bleach. Use of gloves first cleaned by either using 70% ethanol or by changing them frequently is recommended. Use tips and containers that are clean and sterile, such as ATP and nuclease-free consumables. Do not leave reagents or the plate opened while working or during assay incubation Total levels of cellular ATP can be used to assess cell viability, cell proliferation and cytotoxicity of a wide range of compounds and biological response modifiers.We also offer a very popular alternative colorimetric/fluorometric ATP assay kit ab83355based on the phosphorylation of glycerol.Related assaysReview thecell health assay guideto learn about kits to perform acell viability assay,cytotoxicity assayandcell proliferation assay.Review themetabolism assay guideto learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.Abcam has not and does not intend to apply for the REACH Authorisation of customers uses of products that contain European Authorisation list (Annex XIV) substances.It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses. Functional Studies - Luminescent ATP Detection Assay Kit (ab113849)D?ini?, Tamara and Norbert A Dencher., Oxidative medicine and cellular longevity?vol. 2018 7567959., Fig 6, doi:10.1155/2018/7567959 Total cellular ATP concentration. ATP in SH-SY5Y cells cultivated at 21% and 5% O2 24 h after treatment with A? peptide and/or 18 h X-ray irradiation, normalized to cell count, and compared to respective controls. ATP concentration was about 1.3- to 1.8-fold higher at all conditions in cells cultivated at 5% O2 compared to 21% O2. Combination of A? peptide treatment and irradiation resulted in a significantly increased (~1.5-fold) ATP concentration at 5% O2 compared to the control. Samples were measured at least in duplicates (n = 2-4) in three independent experiments (N = 3). Mean SEM analyzed by two-way ANOVA with Tukey\'s multiple comparison test with p The ATP standard curve was prepared as described in the protocol. Background-subtracted data values (mean +/- SD) are graphed. ab113849 ATP detection kit cytotoxicity data. 25000 HepG2 cells were seeded into each well, allowed to adhere and treated for 4 hours with 25µM rotenone and vehicle control (DMSO) in glucose based complete media. After treatment, cells were lysed, exposed to the ATP substrate solution and signal was measured on a luminescent counter. Mean and standard deviation is plotted for 3 replicates from each condition. Rotenone induces cytotoxicity in HepG2 cells. Cellular Energy Flux for HepG2 cells (seeded at 65,000 per well), treated with a combination of drug compounds modulating the ETC (Antimycin A [1 M] and FCCP [2.5 M]), shown as a percentage relative to untreated control cells. Comparative measurements were taken with Extracellular Oxygen Consumption Assay (ab197243) (white column) and Glycolysis Assay [Extracellular acidification] (ab197244) (black column) show the shift between mitochondrial respiration and glycolysis and the cellular control of energy (ATP; measured 1h post-treatment using Luminescent ATP Detection Assay kit (ab113849) (striped column)). Analysis of the release of ATP by connexin hemichannels in stem cells using ATP luminescence kit (ab113849).Cells were cultured in HBSS to induce hemichannel opening. Calcium and GAP-inhibitor were used to trigger hemichannel closure. After two hours the supernatant was collected and ATP was measured according to the protocol (detergent was also applied).Calcium treatment and inhibition by GAP decreased ATP concentration, compared to HBSS control. Graph shows data of three independent experiments. 发表研究结果有使用 ab113849？请让我们知道，以便我们可以引用本数据表中的参考文章。 ab113849 被引用在 111 文献中. Poveda-Huertes D et al. An Early mtUPR: Redistribution of the Nuclear Transcription Factor Rox1 to Mitochondria Protects against Intramitochondrial Proteotoxic Aggregates. Mol Cell 77:180-188.e9 (2020).PubMed: 31630969 Choi J et al. Comparative analysis of the mitochondrial morphology, energy metabolism, and gene expression signatures in three types of blastocyst-derived stem cells. Redox Biol 30:101437 (2020).PubMed: 31981893 Zhang S et al. Nuclear bodies formed by polyQ-ataxin-1 protein are liquid RNA/protein droplets with tunable dynamics. Sci Rep 10:1557 (2020).PubMed: 32005838 Broniarek I et al. The Influence of Statins on the Aerobic Metabolism of Endothelial Cells. Int J Mol Sci 21:N/A (2020).PubMed: 32098258 Somasekharan SP et al. G3BP1-linked mRNA partitioning supports selective protein synthesis in response to oxidative stress. Nucleic Acids Res N/A:N/A (2020).PubMed: 32406909 View all Publications for this product This kit is very simple and easy to use.Always had consistent results.I highly recommend this kit. The kit was used to analyze changes in ATP levels in BV2 microglia cells. The cells were treated with 1µM lysophosphatidic acid (LPA) for the indicated time points. Rapid increase in the levels of ATP were observed at short time points. The kit was easy to use and get the results in less than an hour. We observed some variation between triplicates but did not affect much the outcome. The kit is very easy to use and the results are reproducible. We used it to evaluate changes in ATP levels in primary murine microglia (in BV2 cell line as well) after treatment with various concentrations of lysophosphatidic acid (LPA). We have analyzed the release of ATP by connexin hemichannels in stem cells using the ATP luminescence kit (ab113849).Cells were cultured in HBSS to induce hemichannel opening. Calcium and GAP-inhibitor were used to trigger hemichannel closure. After two hours the supernatant was collected and ATP was measured according to the protocol (we also applied the detergent). As expected calcium treatment and inhibition by GAP decreased ATP concentration, compared to HBSS control. Graph shows data of three independent experiments.In summary, we can recommend the ATP luminescence kit (ab113849) for measuring extracellular ATP concentrations. I enrolled to an Abcam trial to check the suitability of this ATP luminescence kit (ab113849) to detect extracellular ATP levels in cancer cells. I personally recommend this kit to measure extracellular ATP levels for its ease of use and for the kindly attention and help I received from the Abcam scientific support specialists. When measuring extracellular ATP levels, it is important to avoid light exposure as much as possible and measure the supernatant without FBS and without phenol-red.Brief description of the protocol: I seeded 50.000 murine breast-to-brain cancer cells in duplicates or triplicates in 96-well-plate. Once they are attached I starve them in FBS-free and phenol red-free media.24 hours after starving I collected the supernatant (100 ul) and transfer it to a white opaque plate. There I followed the protocol proposed in the booklet with only one difference: I do not use detergent. Using the same conditions (cell line, passage, starving time and machine) I repeated 3 times with the same cancer cell line and the results I got are 12,89 nM ATP, 12,30 nM ATP and 7,3 nM ATP. Regarding the conditions I use, I repeated this procedure several times and I chose this condition (24-hours starvation in phenol red-free and serum-free media) because if I starve them for less hours the luminescence units I get from my samples are super low. If I starve them with HBBS overnight the cells look ugly the following day. Moreover I also tried to add detergent in the media but then the RLU I got were more variable in the same biological replicate. There is also higher variability of RLU if I use media with FBS. I did not try to use media with phenol red because I know it interferes with the luminescence readout. To sum up, I got less variability when I starve them for 24 hours with DMEM without FBS and without phenol red. I have looked into this further and can confirm that in testing clear plates have been used with no major issues. However if you do have black plates, that may be more optimal. Read More We haven’t addressed this question experimentally with the reagents.  The answer is probably.  The lysis reagent is denaturing so bound ATP may be made available to the luciferase. This would be difficult to be definitive and different ATP binding proteins may behave differently. Read More In theory it should work, but we don’t know for sure as we have not tested it. Therefore, if you are willing to test and publish the data in the form of review on our website then I would like to invite you to our Abtrial programme. Read More Thank you for your enquiry.Luminescence does not have multiple wavelengths. In Luminescence mode, the instrument simply measures the emission of visible light from the well. What is key in the measurement is the PMT (photomultiplier time). This acts like the aperture of a camera. The higher the number, the more light gets captured. My suggestion is to accept the default PMT settings of the instrument.If working with an opaque plate, you may be able to have lower PMTs which will allow for a lower background and greater sensitivity. Either opaque or transparent plates could be used (as shown in the protocol booklet). The advantage of opaque plates is that it allows for a wider dynamic range.This assay is designed for end-point reading. For development of signal timing, see section C/steps 5 and 6 of the protocol.I hope this will be helpful. If you have any further questions, please do not hesitate to contact me. Read More Thank you for contacting us.The cells do not need to rest or adhere for this assay, you can measure right away. Read MorePlease note: All products are FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES For licensing inquiries, please contact partnerships@abcam.com