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

21-30 of 41 Abreviews or Q&A

Answer

Generally at both time points the ATP readings will be nearly the same. However, there is a slight possibility that Data A might be slightly higher than Data B, albeit the 25% difference sounds too large. This might partially be due to some of the cells still getting lysed and making the solution in the wells slightly unclear for the light to pass through, which leads to a lower reading of Data B than Data A.

Read More

Answer

Vielen Dank für Ihren Anruf.

Hier ist die Antwort aus unserem Labor:

"This is very common question.


The Data A is for the ATP generated by the cells when they are incubated with the NRB.

Data B is for the total ATP released and present in the sample which keeps rising for a few minutes after the cells are incubated with the NRB and finally plateau off.

Before the ADP converter is added you will not know the levels of ADP, since only ATP will be recognized luminometrically. The ADP has to be converted to ATP for recognition.

Data C is after conversion of ADP from the samples to ATP.

Hope this information has been helpful for you.

Please let me know if you have any other questions."

Ich werde allerdings noch einmalbezüglich Ihrer zweiten Frage nachhacken.






Ich hoffe, dies hilft Ihnen erste einmal weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

Benutzen Sie unsere Produkte? Schicken Sie uns einen Abreview. Verdienen Sie sich eine Belohnung!
https://www.abcam.com/abreviews

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Answer

The aim of the two readings is to check if there is a difference between the first reading and the second reading, A and B. They should be the same. If B is generally higher, then ATP signal is still developing, and it seems to make sense to use that reading for the ADP/ATP ratio. I will update you if I learn more from the laboratory that developed the kit.

For comparison of values across multiple plates, it will be best to include replicates of a standard in each plate, for instance, duplicate or triplicate wells of sample X in each plate, to see if there are any large differences. However, the suggested incubation times are minimums and the signals should hit a plateau within those times, so longer incubation for some of the plates should not be a problem.

Read More

Answer

Thank you for your reply.


Unfortunately, we have not specifically tested the kit with bacterial samples, so we are unable to say for sure whether or not additional modifications to the protocol are necessary.


Regarding the use of a spectrophotometer, the kit was specifically designed for detection using a luminometer and thus we cannot recommend using a spectrophotometer.

Read More

Answer

Thank you for contacting Abcam and for your interest in ab65313.


While this kit has not been specifically tested with bacterial samples, we believe that it should be compatible. Be sure to lyse cells in the Nucleotide releasing buffer, per the kit protocol.


I hope this information is helpful. Please do not hesitate to contact us if you have any additional questions.

Read More

Answer

Thank you for contacting us.
I am sorry to hear you have been experiencing problems to dissolve the ATP enzyme. Could you please provide the order number?
As this product is covered by our Abpromise guarantee I will be happy to send a replacement in compensation for the troubles caused.
I look forward for your reply. Please do not hesitate to contact us for further assistance.

Read More

Answer

Thank you for your questions. My colleague is out of the office today but I would like to forward the response that we received from the lab:

Unfortunately, these three kits have different Assay buffers and converting enzyme mixes, so they can not be shared.

I hope this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

Read More

Answer

I'm sorry for the delay in getting back to you.

I have consulted with the lab over the protocol you have been using and the data you have observed with theADP/ATP Ratio Assay Kitbut we have not beenable to come up to any obvious reasons why the data is not as expected. It may unfortunately be that one of the components of the kit has degraded for some reason.Whilst this is rare, it does notdiminish the inconvenienceit has caused you. I would like to offer you a free of charge replacement of the kit. If you would prefer I can also offer a credit note or refund.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

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Answer

I have some more details that I think will help:

Data A = ATP level
Data B = ATP background level
Data C = ATP background level + ADP converted to ATP

Protocol clarification:

RE: Step 3 - Add 100 ul reaction mix to an empty well in the reaction plate (plate that will be placed in luminometer, not the plate the cells are growing in) for each sample and control well.

RE: Step5 - For adherent cells, remove culture medium and treat cells (103 – 104) with 50 μl of Nucleotide Releasing Buffer for 5 minutes at room temperature with gentle shaking. Nucleotide Releasing Buffer will lyse the cells. Transfer 10ul into the corresponding well containing the 100ul reaction mix in the luminometer plate.

RE: Step 7 - To measure ADP levels in the cells, read the samples (from step 6) again 10 min after the initial reading (Data B). Then add 1 μl of ADP Converting Enzyme and read the samples 1 min later in a luminometer (Data C).

I hope this information is helpful to understand the protocol. Please let me know if you have any further questions.

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21-30 of 41 Abreviews or Q&A

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