Product Details
| abID | ab65314 |
|---|---|
| Detection Method | Luminometer or Beta Counter. |
| Species Reactivity | Mammalian |
| Applications | Bioluminescent detection of the ATP level via luciferase catalyzed reaction for a rapid screening of apoptosis and cell viability in mammalian cells. |
| Features & Benefits | • Simple one-step procedure; takes only 30 minutes • Fast and convenient • The assay can be done directly in culture plates requiring no harvest/washing/or sample preparations. The assay can be fully automatic for high throughput (10 seconds/sample) and is highly sensitive (detects 10-100 mammalian cells/well). |
| Kit Components | • Nucleotide Releasing Buffer • ATP Monitoring Enzyme • Enzyme Reconstitution Buffer • ATP (MW 551) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Cell death (especially apoptosis) is an energy-dependent process that requires ATP. As ATP levels fall to a point where the cell can no longer perform basic metabolic functions, the cell will die. A typical apoptotic cell exhibits a significant decrease in ATP level. Therefore, loss of ATP level in cell has been used as an indicator of cell death. In contrast, cell proliferation has been recognized by increased levels of ATP. The ApoSENSOR™ Cell Viability Assay Kit utilizes bioluminescent detection of the ATP levels for a rapid screening of apoptosis and cell proliferation simultaneously in mammalian cells. The assay utilizes luciferase to catalyze the formation of light from ATP and luciferin, and the light can be measured using a luminometer or Beta Counter. The assay can be fully automatic for high throughput (10 seconds/sample) and is extremely sensitive (detects 10-100 mammalian cells/well). The high sensitivity of this assay has led to many other applications for detecting ATP production in various enzymatic reactions, as well as for detecting low level bacterial contamination in samples such as blood, milk, urine, soil, and sludge.
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Why did my ATP Monitoring Enzyme completely not 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.
Can either the cell viability assay and LDH-Cytotoxicity Assay Kits be used to measure the inhibition of lysis instead of direct lysis?
The kit K254 measures cell viability by monitoring ATP levels; cells are lysed in the Nucleotide releasing buffer and the ATP levels are then analyzed. The kit K311 measures the cell death by monitoring the LDH levels in the cell culture supernatant. “LDH is a stable cytoplasmic enzyme present in all cells and rapidly released into the cell culture supernatant upon damage of the plasma membrane.” I think that the kit K311 will be more appropriate in your case.
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.
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 kit's 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 kit's 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, I would strongly recommend you 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.
Hani N. Sabbah, Effects of elamipretide on skeletal muscle in dogs with experimentally induced heart failure. ESC Heart Fail, Jan 2019; 30688415.
Fujisawa et al., Chronic Hyponatremia Causes Neurologic and Psychologic Impairments. J. Am. Soc. Nephrol., Sep 2015; 10.1681/ASN.2014121196.
Wilk et al., Molecular Mechanisms of Fenofibrate-Induced Metabolic Catastrophe and Glioblastoma Cell Death. Mol. Cell. Biol., Jan 2015; 35: 182 - 198.
Alavian et al., The Mitochondrial Complex V–Associated Large-Conductance Inner Membrane Current Is Regulated by Cyclosporine and Dexpramipexole. Mol. Pharmacol., Nov 2014; 87: 1 - 8.
Paczkowski et al., Fatty acid metabolism during maturation affects glucose uptake and is essential to oocyte competence. Reproduction, Sep 2014; 148: 429 - 439.
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