Overview

  • Product name

    ADP/ATP Ratio Assay Kit (Bioluminescent)
  • Detection method

    Luminescent
  • Sample type

    Tissue, Adherent cells, Suspension cells
  • Assay type

    Semi-quantitative
  • Sensitivity

    < 100 cells/well
  • Assay time

    0h 30m
  • Product overview

    ADP/ATP Ratio Assay Kit (Bioluminescent) ab65313 is based on the bioluminescent detection of ADP and ATP levels. It can be used for a rapid screening of apoptosis, necrosis, growth arrest, and cell proliferation simultaneously in mammalian cells.


    In the ADP/ATP assay protocol, luciferase catalyzes the conversion of ATP and luciferin to light, which in turn can be measured using a luminometer or Beta Counter. The ADP level is measured by its conversion to ATP that is subsequently detected using the same reaction.


    The assay can be fully automatic for high throughput and is highly sensitive (detects 100 mammalian cells/well).


    ADP/ATP Ratio assay protocol summary:
    - transfer suspension cells, or nucleotide releasing buffer treated adherent cells, to plate
    - add ATP reaction mix and incubate for 2 min
    - analyze with luminescence plate reader to measure ATP
    - after preparing ADP reaction mix and measuring luminescence levels again, add ADP reaction mix to same wells and incubate for 2 min
    - analyze with luminescence plate reader to measure ADP

  • Notes

    Changes in the ADP/ATP ratio have been used to differentiate the different modes of cell death and viability. Increased levels of ATP and decreased levels of ADP have been recognized in proliferating cells. In contrast, decreased levels of ATP and increased levels of ADP are recognized in apoptotic cells. The decrease in ATP and increase in ADP are much more pronounced in necrosis than apoptosis.

    Related assays

    Review the cell health assay guide to learn about kits to perform a cell viability assaycytotoxicity 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.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components Identifier 200 tests
    ADP Converting Enzyme (Lyophilised) Blue Cap 1 vial
    ATP Monitoring Enzyme (Lyophilised) 1 vial
    Enzyme Reconstitution Buffer 1 x 2.15ml
    Nucleotide Releasing Buffer 1 x 50ml
  • Research areas

  • Relevance

    The changes in ADP/ATP ratio have been used to differentiate the different modes of cell death and viability. Increased levels of ATP and decreased levels of ADP have been recognized in proliferating cells. In contrast, decreased levels of ATP and increased levels of ADP are recognized in apoptotic cells. The decrease in ATP and increase in ADP are much more pronounced in necrosis than apoptosis.

Images

  • Quantitation of glutamate levels (A) using ab138883 and intracellular ATP (B) using ab65313 in D. hydrothermalis cells grown under different pressure conditions.

  • ADP/ATP ratio in cystathionine-beta-synthase slienced A2780 (Ovarian cancer cell line) cells and AOAA (aminooxyacetic acid) treated A2780 cells were measured using ADP/ATP ratio assay kit (ab65313).

     

  • ADP/ATP ratio were examined in both narciclasine (ncls) and vehicle (veh) treated C2C12 myotubes with or without PA treatment using ADP/ATP ratio assay kit (ab65313). 

Protocols

References

This product has been referenced in:

  • Korski KI  et al. Hypoxia Prevents Mitochondrial Dysfunction and Senescence in Human c-Kit+ Cardiac Progenitor Cells. Stem Cells 37:555-567 (2019). Read more (PubMed: 30629785) »
  • Whang YM  et al. Rapamycin enhances growth inhibition on urothelial carcinoma cells through LKB1 deficiency-mediated mitochondrial dysregulation. J Cell Physiol 234:13083-13096 (2019). Read more (PubMed: 30549029) »
See all 48 Publications for this product

Customer reviews and Q&As

31-40 of 41 Abreviews or Q&A

Answer

Thank you for contacting us and your interest in our products.

I am sorry to hearthat you have been having problems with this kit. I have had a look into the problem that you have been describing and nothing is immediately evident to be causing this problem. If you wouldn’t mind answering the following questions it may be that in understanding the protocol you are using more fully, I can identify what may be going wrong.

SAMPLE

What cells are you using to perform this experiment?
How are you inducing apoptosis?
How many cells are used for every experiment?
Are they cultured as a suspension or as adherent cell culture?



PROCEDURE

Was the nucleotide releasing buffer allowed to equilibrate to and used at room temperature?
Were the enzymes kept on ice and protected from the light prior to use?
What quantity of ATP monitoring enzyme and nucleotide releasing buffer added?
How much ADP converting enzyme was added to each sample?



MEASUREMENT

How was the measurement taken? Fluorescent, Luminescence or Colorimeter? And at what wavelength?
Which kind of plate was used?



STORAGE

How was each of the constituents stored following reconstitution?



ADP Converting Enzyme
ATP Monitoring Enzyme
Enzyme Reconstitution Buffer
Nucleotide Releasing Buffer



DATA

If you could provide mewith the data you have been observing that would be very useful.

Hopefully with thisadditional information I willbe able tounderstand the problem encountered morefully. If you could also provide me with the order number or lot number of the kit, I willbe able to check if we have hadany lot relatedproblems reported withthis kit.

I look forward toreceiving your reply.

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Question
Answer

Thank you for contacting us. After checking with several of my colleagues, the standard curves for our ATP (ab83355) and ADP (ab83359) kits would be compatible with the ADP/ATP ratio kit ab65313. Unfortunately, we are unable to add those standards to our catalog for purchase separately.

We sell ATP disodium salt (ab120385), which we have not tested for use with this kit, but would theoretically be suitable for use as a standard. I hope this helps, please let me know if you need any additional information.

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Question
Answer

Thank you for contacting Abcam. After having talked to the lab, they say theoretically this kit should also work with plant cells as long as there is active ATP in the cell lysate. The freshly prepared cell lysate can be diluted in the Nucleotide Releasing Buffer and then processed in a similar manner. Some optimizations will have to be made to use the plant samples with this kit, but there is a good chance that it will work. Please let me know if you have any more questions.

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Answer

Thank you for your enquiry. Theoretically since the key components are lyophilized, the chances that the kit will still work and I would consider trying it. However, it is possible that some of the components may have been affected, and regrettably we would no longer be able to provide our Abpromise guarantee that it would work. I hope this will be helpful to you. I wish the customer good luck with their experiments, and if you have any further questions please do not hesitate to contact us.

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Answer

Sorry for the confusion here. I did receive a reply from the sourcing lab regarding this question. Unfortunately the extraction buffer constituents are considered proprietary and I have been unable to obtain even the slightest detail regarding what aspect of the extraction buffer may be effecting your assay. I'm sorry I can't be of further assistance in this instance. Please let me know if you have any further questions.

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Answer

The answers are as follows:  1. It is best to use the samples immediately, given the very sensitive nature of these metabolites. 2. Yes you may use the sample to measure total protein. 3. The nucleotide releasing agent does not contain protease or phosphatase inhibitors but you may add these inhibitors for your studies. Optimizations may have to be performed.   I hope this is helpful. Please contact us again if you have any further questions.

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Answer

1. Kit ab65313 should be suitable for your samples but this also depends on the amount of ATP in your samples. 2. Data A is ATP level. Data B is ATP background. Data C is ATP background + ADP converted to ATP. ADP converting enzyme convert ADP to ATP. The 10 min is let the ATP background stablize before converting ADP to ATP. I would suggest to carry out the protocol as recommended when using this kit. The other kits we have are for either ATP or ADP detection separately (reactions in 30min).

Read More

Answer

If the cells samples are fresh and handled immediately after harvesting as indicated in the data sheet, deproteination is not essential with this kit. With tissue samples it may still not be necessary to de-proteinate, although this may vary sample to sample. You might initially try without de-proteination but choose to use the 10kDa MW spin columns (ab93349) to de-proteinate if your data has a large variance, experiment to experiment. I hope this is helpful. Please contact us again if you need any further information.

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Answer

Thank you for your enquiry. I am pleased to help answer your queries about this kit: 1. The procedure can be done using explant tissue samples as well. The samples will need to be homogenised and lysed in the following manner: Start with 10-100 mg of the tissue, add 500-1000 μl (or ~2-3 volumes) of the nucleotide releasing buffer on ice, homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Load the supernatant unto a 10 kda spin column for deproteinization (if indicated in the data sheet; not required for enzyme assays). Use the eluate for your subsequent assays. Appropriate dilutions of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve. 2. Apoptosis can be induced using any conventional method. 3. Yes the kit is suitable for explant tissue as long as the tissue sample will be homogenized using the Dounce homogenizer the samples will be lysed. 4. There should be 10 minutes between reading Data A and Data B. I hope this will be helpful to you. Should you have any further questions, please do not hesitate to contact us.

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Answer

This kit has not specifically been tested with bacterial samples, theoretically though this kit should be compatible with these samples if they are lysed in Nucleotide releasing buffer as indicated in the specific protocol for this product.

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31-40 of 41 Abreviews or Q&A

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