ATP Assay Kit (Colorimetric/Fluorometric) (ab83355)

Reviews (5)Q&A (32)References (130)
ATP levels in Pancreatic Islet
  • ATP assay used to study mitochondrial disfunction after C. rodentium infection in mice
  • ATP assay performed with ab83355
  • ATP assay performed with ab83355
  • ATP assay performed with ab83355
  • ATP levels measured in mouse colon tissue
  • ATP assay performed with ab83355
  • ATP assay performed with ab83355

Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric/Fluorometric
  • Platform: Microplate reader
  • Assay time: 1 hr
  • Sample type: Cell Lysate, Other biological fluids, Plasma, Serum, Tissue Extracts, Urine
  • Sensitivity: 1 µM

Overview

  • Product name

    ATP Assay Kit (Colorimetric/Fluorometric)
    See all ATP kits

  • Detection method

    Colorimetric/Fluorometric

  • Sample type

    Urine, Serum, Plasma, Other biological fluids, Tissue Extracts, Cell Lysate

  • Assay type

    Quantitative

  • Sensitivity

    < 1 µM

  • Assay time

    1h 00m

  • Product overview

    ATP Assay Kit (Colorimetric/Fluorometric) (ab83355) use a robust, simple method; the ATP assay protocol relies on the phosphorylation of glycerol to generate a product that is easily quantified by colorimetric (ODmax = 570 nm) or fluorometric (Ex/Em = 535/587 nm) methods.


    This kit can detect as low as 1 µM of ATP in various samples.


    ATP assay protocol summary:
    - add samples (deproteinized) and standards to wells
    - add reaction mix and incubate for 30 min at room temp
    - analyze with microplate reader


    Chinese protocol available. See protocols section below.


    If you require a more sensitive product, we recommend Luminescent ATP Detection Assay Kit (ab113849), which can detect as low as 1 pM of ATP.

  • Notes

    Related assays

    Review the cell health assay guide to learn about kits to perform a cell viability assay, cytotoxicity assay and cell proliferation assay. 

    Review the metabolism assay guide to 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.

    How other researchers have used ATP Assay Kit ab83355

    This ATP assay kit has been used in publications in a variety of sample types, including:
    - Human: cell culture lysates1, primary monocyte cell culture lysates2, HCT116 cell culture supernatants3
    - Mouse: heart tissue4, liver5, C2C12 and L929 cell lysates6, primary thymocyte cell culture lysates7, cardiac tissue8
    - Rat: primary hippocampal neuron cell culture lysates9, liver tissue10, skeletal muscle11
    - Pig: kidney cell culture lysates12, heart tissue13 
    - C elegans tissue14
    - Chlamydomonas reinhardtii algae15

     

    References: 1 - Civallero M et al 2017, Na JY et al 2018, 2Gkirtzimanaki K et al 2018, 3Yang et al 2018; 4 - Singh SP et al 2018; 5 - Han SJ et al 2018; 6 - Alhindi Y et al 2019, Gregorczyk KP et al 2018; 7 - Simula L et al 2018; 8 - Litt MJ et al 2017, Samokhvalov V et al 2018; 9 - Zhao X et al 2018;  10 - Jing R et al 2018; 11 - Trinchese G et al 2018; 12 - Zou X et al 2018; 13 - Yuan F et al 2018; 14 - Pandey et al 2018; 15 - Ramanan R et al 2018

  • Platform

    Microplate reader

Properties

Associated products

Images

  • ATP levels in Pancreatic Islet
    ATP levels in Pancreatic IsletImage courtesy of Mrs. Fotini Mouth

    Ab83355 was used to determin ATP levels in rat pancreas islets as an ischemic marker to predict transplantation outcomes. We extracted ATP from fresh pancreas that have undergo different time of cold ischemia : 0, 2, 4, 6, 8 and 10h and in situ. ATP were extracted in Perchloric acid (PCA-2M) and grind using a Polytron. PCA were removed using potassium hydroxide (KOH – 2M) and pH was adjust around 7-8. Samples were conserved at -80°C before utilization.

  • ATP assay used to study mitochondrial disfunction after C. rodentium infection in mice
    ATP assay used to study mitochondrial disfunction after C. rodentium infection in miceImage courtesy of Maiti A K et al. PLoS One. 2018; 13(9): e0204567. doi: 10.1371/journal.pone.0204567

    Maiti AK et al (2018) used ATP assay kit ab83355 to measure mitochondrial ATP generation in an in vitro mouse intestinal model treated with cytokines in the presence and absence of VIP (vasoactive intestinal peptide). VIP was induced by C. rodentium infection and cytokines.

  • ATP assay performed with ab83355
    ATP assay performed with ab83355Sanokawa-Akakura R et al., PLoS One 9(9), fig4b. doi: 10.1371/journal.pone.0108537 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    The chart shows a comparison of ATP levels of HepG2 treated with 0, 10 and 100 µM for 48 hours, DRH2O2 W1 (damage recovered cells using hydrogen peroxide with a recovery time of one week) HepG2 cells and MDA-MB-231 cells treated with 0, 10 and 100 µM of NaHS for 48 hours. Data is shown as percent of ATP levels in untreated cells. ATP levels were determined using ATP assay kit (ab83355).

  • ATP assay performed with ab83355
    ATP assay performed with ab83355Image from Shi Z et al., PLoS One 9(8), Fig 2 doi: 10.1371/journal.pone.0105675. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    MCF-7 cells are transfected with vector, osteopontin-a, osteopontin-c or osteopontin -a plus -c. Cells are plated on poly-HEMA and seeded at 4 x 105 cells per well and incubated for two days under standard culture conditions. ATP levels are measured using ATP assay kit (ab83355).

  • ATP assay performed with ab83355
    ATP assay performed with ab83355
    Example of fluorometric ATP assay standard curve.
  • ATP levels measured in mouse colon tissue
    ATP levels measured in mouse colon tissueImage courtesy of Maiti A K et al. Sci Rep. 2015; 5: 1543. doi: 10.1038/srep15434. Reproduced under the Creative Commons License http://creativecommons.org/licenses/by/4.0/

    Maiti AK et al. (2015) used ATP assay kit ab113852 to measure mitochondrial ATP generation in murine distal colon after C. rodentium infection.

  • ATP assay performed with ab83355
    ATP assay performed with ab83355
    Example of colorimetric ATP assay standard curve.
  • ATP assay performed with ab83355
    ATP assay performed with ab83355

    Quantitation of ATP in fish liver (2.5µl of 10 times diluted sample), fish muscle (5µl of 10 times diluted sample) and Jurkat cell lysate (5 ul) using fluorometric assay. Samples were spiked with known amounts of ATP (300pmol).

References (130)

Publishing research using ab83355? Please let us know so that we can cite the reference in this datasheet.

ab83355 has been referenced in 130 publications.

  • Udi S  et al. Dual inhibition of cannabinoid CB1 receptor and inducible NOS attenuates obesity-induced chronic kidney disease. Br J Pharmacol 177:110-127 (2020). PubMed: 31454063
  • Lee YJ  et al. Down-regulation of the mitochondrial i-AAA protease Yme1L induces muscle atrophy via FoxO3a and myostatin activation. J Cell Mol Med 24:899-909 (2020). PubMed: 31725201
  • Phan K  et al. Uncovering pathophysiological changes in frontotemporal dementia using serum lipids. Sci Rep 10:3640 (2020). PubMed: 32107421
  • Guan Y  et al. Circular RNA circPITX1 knockdown inhibits glycolysis to enhance radiosensitivity of glioma cells by miR-329-3p/NEK2 axis. Cancer Cell Int 20:80 (2020). PubMed: 32190004
  • Sarkar TJ  et al. Transient non-integrative expression of nuclear reprogramming factors promotes multifaceted amelioration of aging in human cells. Nat Commun 11:1545 (2020). PubMed: 32210226
View all Publications for this product

Customer reviews and Q&As

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1-10 of 37 Abreviews or Q&A

Question

Can you please let me know if desferrioxamine interferences with the ATP assay?

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Answer

Unfortunately, we have not tested the interference of desferrioxamine with this kit.

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Question

Do you have any recommendations for the type of 96 well plate to use for the fluorometric assay? Black, flat bottomed, low/high binding?

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Answer

For the colorimetric assay, use a clear plate. For the fluorometric assay a clear or a white plate can be used. Use flat bottom plates.

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Question

I want to use ATP Assay Kit to determine the ATP level in bone marrow cells. My question is the ATP level measured by this kit is total ATP (cellular plus inside mitochondria) or only cellular ATP?

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Answer

This kit will detects total ATP.

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Question

The kit instructions are pretty vague on the cell lysis step.  Can you advise us on a methodology for lysing tissue in the ATP Assay Buffer? Specifically for how long, and at what temperature?

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Answer

There are 2 ways you can efficiently lyse your cells for this assay kit. You can either homogenize the 1 x 10^6 cells with 100 ul of the assay buffer using 30-50 passes on ice, check for the homogenization and then deproteinate your samples with the deproteinization kit. Alternatively, you can just lyse your cells in the PCA using the protocol mentioned for the deproteinization kit, neutralize the lysate and use it in the wells. In this case you will have to use the assay buffer for diluting the samples in the wells.

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Question

Should protease inhibitors be added to step 1 of the sample prep (lysing cells in ATP assay buffer)?

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Answer

Yes, protease inhibitors can be added during step 1 of the sample preparation step.

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Question

Your protocol mentions that the ATP standard vial is 1 micromol; how can you can make a 10 mMOL stock from 1 Micromol? As you mentioned in page 7 1/100 dilution of 1m MOl (1000 Micro molar or 1000000 nano molar) generates 0,2,4,6,8,10 micro molar not nano molar?

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Answer

The calculations are as follows;

1umol is not micro mole means, it is not molarity it is absolute weight.

1micromol is dilutes in 100ul of water which will give you 1micromol/100ul or 1/100 micromol per ul

Now 10ul of this is diluted further 10 time i.e. 1/100x1/10 = 1/1000 micromol/ul which means 1nanomol/microlitre

1ul = 1 nanomol

2ul = 2 nano mol

10ul = 10 nanomol

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ab83355 for zebrafish larvae (80 hours post fertilization)

 Poor Excellent 5/5 (Ease of Use)
Abreviews
abreview image
We tested the kit ab83355 to quantify the total ATP in 80 hours post fertilization zebrafish larvae. ATP was quantified by fluorometric method (Ex/Em = 535/587 nm) using a TECAN Infinite 200 PRO.
Ten l0 hours post fertilization (hpf) larvae were prepared following the protocol given by Abcam. Two technical replicates were run in parallel with a background control (mix without ATP converter).

According to our results, the background noise generate by glycerol phosphate does not allow the quantification of ATP in the larvae. Additionally, the kit seems to be not sensitive enough to detected ATP in 80 hpf zebrafish larvae. In fact, all measured values are below the last point of the calibration curve.

We tried different experimental set ups:
Fig. 1: Deproteinization using PCA (45 minutes) as described by the Abcam protocol. Each bar represents a biological replicate. 50 μl of sample was tested.
Fig. 2: Different deproteinization times (5 and 1 minute) and no deproteinization step. 25 μl of sample was tested.
Fig.3: Different dilutions (10 larvae in 100 μl buffer and 50 μl buffer) and clean-up without using acids (chloroform clean-up). 40 and 30 μl of sample were tested, respectively. Experiments were performed in duplicates.

Despite we tried different dilutions, deproteinization and clean-up steps, the general low concentration of ATP and the strong background noise does not allow the quantification of ATP in the larvae. Consequently, we do not recommend to use this kit for zebrafish larvae.
However, we would happy to try a more sensitive methods such as the Luminescent ATP Detection Assay Kit (ab113849).

Mr. Riccardo Massei

Verified customer

Submitted Oct 14 2020

CYP-450 Epoxygenase Derived Epoxyeicosatrienoic Acid Contribute To Reversal of Heart Failure in Obesity-Induced Diabetic Cardiomyopathy via PGC-1 α Activation

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
I tried many different products to get better ATP measurements in Heart tissue of mice but failed then I bought abcam ATP Assay Kit (Colorimetric/Fluorometric) (ab83355) to measure ATP content in heart tissue of mcie which was knockout to PGC1 alpha, as recommended in their datasheets and I never had any problem with it. I got very important data which I used for my research article. I think its great kit to use and have everything in it. I think this is best product to detect ATP content.

Abcam user community

Verified customer

Submitted Aug 07 2018

Testing ab83355 kit in cell culture media

 Good Excellent 5/5 (Ease of Use)
Abreviews
abreview image
We are interested in cuantifying the amount of ATP released to the culture media from primary astrocytes and microglia BV2 after their treatment with different compounds. To that end, we collected the media (DMEM with 1% of penicilin/streptomicin and 4mM of L-glutamine, but without phenol red) and froze them in liquid nitrogen. When we proceeded with the quantification, we thawed the samples on ice and centrifuged them at 13,000 xg for 10 minutes, without applying any deproteinization step. After this, we went on with the assay. We tested the two methodologies available: colorimetric and fluorimetric. Using cells as positive control, we obtained signal by both procedures, but fluorimetric's was the most sensitive, with the hightest signal/background ratio.

Abcam user community

Verified customer

Submitted Jul 31 2018

ATP measurement after siRNA treatment in cancer cells

 Excellent Excellent 5/5 (Ease of Use)
Abreviews
abreview image
We knockdown a protein which we are studying to effect the cell proliferation.
siRNA knockdown showed significant reduction in ATP generation in cancer cell lines.

Abcam user community

Verified customer

Submitted Mar 27 2018

1-10 of 37 Abreviews or Q&A

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