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

11-20 of 41 Abreviews or Q&A

Answer



1. If you are using adherent cells, than yes, you will get a total volume of 150ul.

2. Will have to ask the lab for this, as it not clear from the protocol if more or less buffer is needed. I will get back to you as soon as I receive an update.

3. Please contact our official distributor in Australia, Sapphire, regarding the price and delivery time.

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Question
Answer

Vielen Dank für Ihren Anruf.

Ich habe nun Nachricht vom Labor erhalten mit mehr Informationen über die Verarbeitung von Gewebe für dieses Kit:

Es muss eine Single-Cell-Suspension aus Gewebe hergestellt werden.

Die Einzelzellen werden dann wie adhärente Zellen im Protokoll behandelt.

Es kann auch versucht werden des Gewebe so klein wie möglich zu zerkleinern/schneiden und dann 5 Minuten bei Raumtemperatur in Nucleotide releasing buffer inkubieren.

Die Vorbereitung von Gewebe muss bei diesem Kit immer optimiert werden, da eine erhöhte Anzahl toter Zellen das Ergebnis beeinflussen kann.

Es tut mir leid, dass ich keine präzisere Antwort für Sie habe und hoffe, diese Information war dennoch hilfreich.

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Answer

Thank you for contacting us.

No, there are no ATPase Inhibitors in the buffers. The ATP is very labile and hence the reaction must be carried out within the set timeframe. Yes, you can make standard curves with pure ATP/ADP.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer



1) Yes, standard protein quantitation assays can be performed with extracts with the NRS buffer.

2) We have not tested for arsenic influence, but if you are using arsenic only to induce apoptosis, then it should not interfere with the readings or quenching.

3) Yes, a standard curve can be made for which you would need pure ADP/ATP, which is not provided in the kit.

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Answer

Thank you for contacting us.

I can confirm that the ADP/ATP Ratio Assay Kit (Bioluminescent) - ab65313 is guaranteed to work with tissue extracts like heart tissue.

The Kit is likely to work with frozen tissue. However, we recommend to use a fresh sample as control if possible to be able to compare if there are differences due to the snap freezing of samples.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

This kit may be applicable to your studies, but I'd recommend looking in the literature for the best, most widely used way to distinguish between aerobic respiration and glycolysis. Abcam's ADP/ATP Ratio Assay Kit (Bioluminescent) utilizes bioluminescent detection of the ADP and ATP levels for a rapid screening of apoptosis, necrosis, growth arrest, and cell proliferation simultaneously in mammalian cells.

You can find the references for this kit here: https://www.abcam.com/ADPATP-Ratio-Assay-Kit-Bioluminescent-ab65313-references.html

And this article describes a technique for cancer cells: http://www.seahorsebio.com/resources/tech-writing/appnote-phenotypes-cancer-cells.pdf

You may be interested in some of our other metabolism kits:

https://www.abcam.com/index.html?pageconfig=resource&rid=13854

https://www.abcam.com/index.html?pageconfig=resource&rid=13571&source=pagetrap&viapagetrap=assaykits

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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https://www.abcam.com/abreviews

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Answer



Unfortunately, this kit only aims to give you an overview of the ADP/ATP ratio to make a statement about the cell fate (cell viability assay)of your samples.

As this is a Semi-quantitative test, you can only measure if A and/or B is there, and how much more of A is there than B, but youdo not measure units or weight.The latter can only be done by a quantitative measuring.

I hope this information is still helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

I just heard back from the lab with the following advice:

The short life of ATP is very low. If the cells have to be frozen for later ATP analysis, I think there are protocols involving the use of TCA to do so and once thawed this TCA has to be neutralized. Since we have not done this ourselves, we cannot recommend the exact protocol. Therefore the success of this kit depends on how well and under what conditions the customer had frozen the samples.

I hope this information will help somewhat further. But to know for sure, it would need to be tried out. The TCA is to precipitate proteins.




Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

In this assay, it requires 10^4 to 10^6 cells for each reaction. For each 10^3 to 10^4 cells, it needs 50 ul of the nucleotide releasing buffer (NRB) which means that if a maximum 10^6 cells in each sample is used, you will need to add at least 500 ul of NRB to it.

This assay is designed for a 96-well plate, totally 200 assays in the 96-well format. If you would like to use about 2 x 10^5 cells for each sample,you can easily use the 96 well plate. Please be reminded that you are not growing the cells in this plate, just assaying them in it. You can grow the cells in any plate, even a 10 cm plate would do.

As this is a luminiscence assay, you will have to use a 96-well white plate for the analysis. Do not use a 24-well plate because you will have to scale up the reagents by about 4 times which means that only about 50 tests can be performed with this kit or you will need to purchase more reagents to account for the increase in reagent volume.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us.

We do not have an article describing the principle of the assay, but the datasheet includes the following notes, which you may have already seen. There are also six references we know of that have used the kit, and these are also found on the online datasheet.

The assay, product ab65313, utilizes the enzyme luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer or Beta Counter. ADP level is measured by its conversion to ATP that is subsequently detected using the same reaction.

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.

We are not aware of any direct comparison of the kits to the MTT or Trypan Blue assay of cell death.

Trypan Blue will not be able to identify apoptosis as accurately as this kit, since staining cells with Trypan Blue depends on disruption of the plasma membrane, which does not occur until very late in the apoptotic process. Trypan Blue dye is used to identify necrosis, but does not distinguish between late-stage apoptosis and necrosis.

Our distributor in Korea is Dawinbio:

Tel
+82 1588-9741
Website
http://www.dawinbio.com
Fax
+82 317904360
Email
dawinbio@dawinbio.com

If you have any technical questions, please contact us at technical@abcam.com.

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11-20 of 41 Abreviews or Q&A

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