Going with the glow
Luminescence-based assays can be quick and easy to use, as long as you follow these rules.
Luminescence assays are excellent tools to examine gene expression and regulation, immunoassays, and more via flash or glow reactions. A luciferase enzyme catalyzes a two-step oxidation of luciferin to yield light (Figure 1). Flash luminescence reactions generate rapid and measurable light once the prepared sample reaches a high energy excited state, and are highly sensitive. Glow luminescence reactions include enzyme inhibitors, and once excited, the reactions generate stable and measurable light for up to several hours, but are generally less sensitive than flash reactions.
No matter the light intensity preference or overall assay purpose, careful attention to the following factors can boost efficiency and greatly improve luminescence assay results.
Because all wells in a luminescence assay glow at the same time, clear microplates were readily recognized as unsuitable because of the extensive amount of signal crosstalk between wells. In 1982, black and white microplates were introduced by Dynatech (now Dynex Technologies) and quickly used in the early fluorescence microplate readers. Black microplates had a lower background, but white microplates absorbed less signal from the assay. Because of better signal to noise results, black microplates became preferred for fluorescence intensity assays while white microplates, with their greater signal emission and reduced signal absorption, became preferred for luminescence. In luminescence assays, opaque white microplates reflect 10% more of the luminescent signal than black microplates. For certain cellular assays, white-sided microplates with clear bottoms have been developed.
|Figure 1. Oxidation of luciferin using different luciferase. (Source: BioTek Instruments)|
At the same time, white microplates absorb energy from room light and phosphoresce, which can significantly affect final results. When reading luminescence assays in white microplates that have absorbed light energy, readings can mimic drifting as the microplate is read and the phosphorescence slowly dissipates.
The effects of room light exposure in luminescence readings are highlighted in Table 1. A black microplate and a white microplate were filled with equal amounts of deionized water and exposed to normal ceiling-mounted fluorescent lights for 20 minutes, and then read on a Synergy 2 Multi-Detection Microplate Reader (BioTek Instruments, Winooski, Vt.), with a maximum sensitivity setting of 255. Results (Table 1) from the first row (Row A) to the last row (Row H) and across the microplate were not statistically significant in the black microplate. In contrast, results from Row A to Row H and across the microplate in the white microplate are statistically significant and show a decrease in signal as the light energy in the microplate dissipates over time. It is also worth noting the clear difference in average readings when comparing the black and white microplates. This difference is directly attributed to the aforementioned reflection of absorbed light in white microplates and lack of phosphorescence in black microplates.
Phosphorescence in white microplates is easily remedied by storing or incubating the microplate in complete darkness for several minutes prior to reading, also known as dark adapting, so long as the incubation period does not conflict with assay protocols.
A microplate’s material of construction can be important, too. Plastic microplates are commonly used for their low cost and convenience, although polystyrene materials may build up static electricity and adversely contribute to background signal. Even the amount of pigment used in the colored microplates may correlate with crosstalk between wells.
|Reading 1||Reading 2||Reading 3|
|Row A Average||209||322||310|
|Row H Average||318||271||274|
|Average of 96 Wells||266||331||314|
|Row A Average||1256||585||459|
|Row H Average||635||384||499|
|Average of 96 Wells||819||504||518|
Several luminescence readers are available, including dedicated luminometers and multi-mode microplate readers. Regardless of which instrument is most beneficial to individual needs, some settings will require modification for optimal luminescence performance. For example, if the photomultiplier (PMT) gain setting is too low, the sample signal will not be amplified above background noise. Conversely, if the PMT gain setting is too high, the PMT might become saturated and the results will be listed as over the range. If an amplification range is not recommended by the instrument manufacturer, a test control or standard can help to identify the proper linear range.
This control can also be used to normalize assay results from other luminescence assays, as the unique combination of instrument, reagents, and reaction conditions make a direct comparison impossible to achieve no matter how carefully the assay is executed.
Finally, the force of reagent injection is noteworthy for flash type assays. The smaller the injection volume, the more difficult it is to achieve complete mixing within each well, especially if the dispense rate is slow. On the other hand, if a dispense rate is too forceful, it may lead to splashing and cross-contamination. Fortunately, most automatic dispensers also allow for a degree of flexibility.
Assay drift, a notable departure from all expected results, can be a commonly misdiagnosed source of error in luminescent assays as there can be several contributing factors. These factors can be loosely categorized into instrument error, mishandling of reagents and consumables, and human error.
The results of most flash assays do not drift because fluid injection, which initiates the reaction, is immediately followed by reading. Glow assay results can drift downward because the luminescence signal eventually declines. Taking too long to dispense the activating reagent with respect to the reading sequence can lead to reading the last wells on the microplate too late if the duration of the luminescence signal is short. Reversal of dispense-to-read well order can mimic drifting effects across the wells of a microplate. These time-dependent delays can overlap with the decay of the luminescent signal. Efficient and automated liquid dispensing, consistent dispense-to-read order, and integration times appropriate for the assay should minimize any erroneous results.
When troubleshooting luminescence assay drift, a calibrated test plate can be used to confirm or rule out mechanical variations as the cause. This test plate emits a stable luminescence signal; therefore, readout variations would be indicative of luminometer-caused variation while a steady readout could indicate that the drift is not related to the luminometer.
The preparation and storage of assay reagents should receive careful consideration. Since the rate of light emission from luminescent reactions varies as a function of temperature, it is important to allow reagents to equilibrate to their optimal working temperature, often room temperature. If reagents are used prior to reaching an optimal working temperature, they will continue to equilibrate while the microplate is being read. This means that the reagent temperature, and resulting luminescent signal, may be significantly different in the first well compared to the last well.
Additionally, once reagents are diluted or reconstituted from their original forms, they will begin to slowly degrade. This may be of concern for those who do not perform the maximum number of luminescent assays per kit in one setting. If use is anticipated over an extended period of time, the reagents should be frozen after aliquoting into several smaller doses to protect from traumatic freeze/thaw cycles that can diminish reagent activity.
As with any assay, poor technique can invalidate results, resulting in a loss of time and an increase in associated assay costs. For example, when using luminescence assays to measure adenosine triphosphate (ATP), contamination with exogenous ATP from skin, cell cultures, bacteria, or contact with any living cell is completely indistinguishable from a sample’s ATP. ATP contamination from these sources can randomly affect results and this lack of consistency cannot be corrected for with a blank or negative control as if ATP contamination consistently affected all wells. There is no existing method to remove the exogenous ATP once exposed to sample ATP, therefore, its presence can lead to unexpected and randomly higher luminescence values.
Additionally, microbial growth in reagent fluid lines from unsatisfactory instrument maintenance can result in a false positive ATP signal. Performing related luminescence assays, such as ATP and luciferase, without proper decontamination and thorough cleaning between the assay types may lead to inordinately high signals in the initial microplate wells due to reagent reactivity.
Gloves should always be used when handling samples, equipment, and reagents associated with luminescence assays—even when transporting these materials to and from storage. Consumables should be stored in a clean environment, and equipment should be cleaned and contaminated per the manufacturer’s instructions on a regular basis. Additionally, all buffers, solutions and consumables should be sterile and ATP-free, if necessary.
Luminescence-based assays are generally simple to perform, and yield results in minimal time. The overall value of the results and subsequent analysis can be increased by focusing attention on the aforementioned topics in addition to following instruction from assay kit and instrument manufacturers.
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November 1, 2008
Ted Quigley, Applications Specialist, Applications Department
BioTek Instruments, Inc.
Winooski, VT, USA