• Detection method- Luminometer or Beta Counter.
• Sample type- Cell and Tissue lysates, culture media, urine, soil, sludge, plasma and serum, as well as many other biological fluids
• Species reactivity- Mammalian
• Applications- Bioluminescent detection of the ADP and ATP levels for a rapid screening of apoptosis, necrosis, growth arrest, and cell proliferation simultaneously in mammalian cells.
Features & Benefits:
• Simple one-step procedure; takes only 30 minutes
• Fast and convenient
• The ADP/ATP ratio assay offers highly consistent results and with excellent correlation to other apoptosis markers (e.g. TUNEL-based assays and caspase assays). In addition the assay can be fully automatic for high throughput (10 seconds/sample) and is highly sensitive (detects 10-100 cells/well).
• Nucleotide Releasing Buffer
• ATP Monitoring Enzyme
• ADP Converting Enzyme
• Enzyme Reconstitution Buffer
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. The ApoSENSORTM ADP/ATP Ratio Assay kit 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. The assay 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 assay can be fully automatic for high throughput and is highly sensitive (detects 100 mammalian cells/well).
USAGE: For Research Use Only! Not For Use in Humans.
Why didnt my ATP Monitoring Enzyme completely dissolve?
The ATP monitor enzyme is hard to dissolve and the final solution is a yellow-green milky solution (not clear solution). This is normal and it is fine to use it.
What kind of Luciferase is used in this assay?
The Luciferase used was expressed in E. coli using the cloned luciferase gene from the North American firefly, Photinus pyralis.It rapidly loses activity after reconstitution. Customer should either use fresh luciferase or deep freeze immediately after reconstitution.
Why is my 10 minutes reading higher than 1 minutes reading?
The facts that the 10 min value is higher than the 1 min value means that when the lysate settled down, it become more clear for light passing through and thus results in higher readings. (Suggestions: Customer may centrifuge the samples to collect and use only the clear portion of the cell lysate). In addition, using more cells (e.g., 10000 cells/assay) may obtain more reliable results.
Does any wavelength limit need to be set while detecting with luminometer ?
No, unlike fluorometric readings where the emission has a specific wave length for reading the excited molecule, and the excitation has an optimum wave length to excite the molecule, the chemiluminescence works on a different principal. The molecule is present at a high energy level. The substrate breaks that constriction and brings it down to the lower and more stable energy level. The difference in energy is released in the form of light. The luminometer captures this light and measures its intensity. There is no wave length setting in this process.
I would appreciate if you could confirm if this kit has ever been used with a Beckman Coulter LS6500 Multipurpose Scintillation Counter. Would you have any advice on how they could set up the instrument to use with this kit? Would this type of reader be suitable?
The read out of this kit is via luminescence and hence if the indicated equipment is a luminometer, the customer can use it. Theoretically a luminometer and a scintillation counter are different. Conventional scintillation counters can’t be used. However, when Beta Counter is used it should be programmed in the “out of coincidence” (or Luminescence mode) for measurement. So if they have this setting where they can program this, then the beta counter may be used.
I used this kit and have levels of ATP and ADP. Say if i want to present the ATP levels independent of the ratio, which concentration unit or how can i convert the levels to concentration?
To get the exact conc, you would need a standard which is not included in this kit. You can always get the ATP standard form our other kit with the Cat # K254-200-4, but you will have to optimize the production of the standard curve.
The protocol suggested that we need to add 50µl Nucleotide Releasing Buffer to 103-104 adherent cell, Im wondering how I can change the volume of Buffer when I have 1x106 Beas-2B cells.
It is recommended you to take just 10^4 cells for this assay. Note that once you treat your cells with 50µl of the NRB, you are taking only 10 ul in the next step. Therefore it would not make sense to increase the volume of NRB to accommodate more number of cells. If the cell number cannot be taken, I would recommend using ~5 ml of the buffer to keep up the proportion.
Why meassuring the level of ADP/ATP so many times?
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.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kits citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration?
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kits components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, It is recommended to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.