Background
Bioluminomics™
What is Bioluminomics™?
Bioluminomics™ is the science of using the cell's energy source in the form of ATP (adenosine triphosphate) to provide information regarding the cell's cellular and mitochondrial integrity. This, in turn, provides valuable information on:
- Viability
- Cellular functionality
- Proliferation status or ability and potential
- Cytotoxicity
- Cell number
- Apoptotic status
Bioluminomics™ is the ability to measure changes in intracellular ATP (iATP) concentration when it reacts with luciferin and luciferase to produce bioluminescence in the form of light that is measured in a plate luminometer. The information obtained is quantitative and standardized, meaning that results from one experiment or study can be directly compared with others over time. In addition, the quantitative nature of the information allows any bioluminomics™ assay to be validated.
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The Concept of Bioluminomics™
Intracellular ATP is a biochemical marker for multiple readouts. First and foremost, iATP is an indicator of cellular and mitochondrial integrity. If the cell does not produce iATP, it is dead. Thus, the iATP concentration is a metabolic indicator of cell viability and can be a far superior marker of viability than dye exclusion methods, which can produce false positive results.
In addition, when cells are stimulated to proliferate, the iATP concentration increases dramatically. Conversely, in the presence of cytotoxic agents, the iATP will decrease significantly. These changes in iATP concentration correlate directly with the cell's functional status and can therefore be used to measure proliferation or inhibition. Furthermore, the iATP concentration is directly proportional to the number of cells present. Thus, the iATP concentration can be used to measure cell number.
Finally, the onset of apoptosis requires ATP. As a result, when apoptosis occurs, ATP is converted to ADP. The ratio of ADP to ATP provides a measurement of apoptosis.
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The Principle of Bioluminomics™
The principle of bioluminomics™ is simple. As cells proliferate or are inhibited from proliferation by cytotoxic or other agents, the iATP concentration varies proportionately. Some cells, such as hepatocytes, have inherently high iATP levels which would decrease in the presence of inhibitory compounds. After incubation, the cultured cells are lysed and the release iATP acts as a limiting substrate for a luciferin/luciferase reaction. One of the products of the reaction is bioluminescence which is measured as light photons in a plate luminometer.
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Measuring Bioluminescence: The Plate Luminometer
A luminometer is an instrument that detects and measures luminescence. The luminescence can be in the form of a chemical reaction that produces flashes of light. This is called chemiluminescence. When luminescence is produced as a result of a biological reaction, it is called bioluminescence. The light produced is usually in the form of a "glow" that remains stable over a period of time. Plate luminometers can usually measure both "flash" and "glow" forms of bioluminecence. No special filters are necessary to measure luminescence.
Luminometers are manufactured either as multiparameter instruments or as dedicated, individual instruments. A multiparameter instrument will also be capable of detecting absorbance and fluorescence, as well as luminescence. A dedicated luminometer only measures luminescence and usually has greater sensitivity than a multiparameter instrument, although this need not necessarily be the case.
The luminometer used will also depend on the type and throughput of application. For the majority of bioluminomics™ applications, such as stem cell and basic research assays, most cellular thereapeutic assays and veterinary assays, HemoGenix® recommends the Promega GloMax®-96 Multiplate Luminometer. This instrument is simple, easy and fast to use, but demonstrates excellent range and sensitivity. In association with Promega, HemoGenix® is now offering the GloMax®-96 Multiplate Luminometer at a special price. For more information, please see our Special Offer.
For cord blood banking and biobanking where sample sizes can be large and for high throughput toxicity screening, HemoGenix® recommends the Molecular Devices SpectraMax L with SoftMax Pro software, since the process can be automated.
A comparison between these two plate luminometers is shown in the graphs. The comparison shows the results for different bone marrow and umbilical cord blood stem and progenitor cells using HALO-96 Research and HALO-96 PMT. The results indicate that both instrument provide similar results. This is to be expected since both exhibit a similar range and sensitivity. Regardless of which of the two instruments you use for our bioluminomics™ assays, HemoGenix® can provide software templates that will allow the user to obtain all the information required from the first assay they perform.