Eight Considerations for Getting the Best Data from Your Luminescent Assays

The stage is set. You’ve spent days setting up this experiment. Your bench is spotless. All the materials you need to finally collect data are laid neatly before you. You fetch your cells from the incubator, add your detection reagents, and carefully slide the assay plate into the luminometer. It whirs and buzzes, and data begin to appear on the computer screen. But wait!

Bad data
These data are garbage!

Don’t let this dramatic person be you. Here are 8 tips from us on things to watch out for before you start your next luminescent assay. Make sure you’ll be getting good data before wasting precious sample!

1. Use the Right Plates

We recommend using white, opaque-walled assay plates because they reflect light, maximizing the output signal for luminescence assays. However, if you are multiplexing a luminescence assay with a fluorescence assay, a white plate can result in higher crosstalk and background for the fluorescence portion of the assay.

Using a black plate in a multiplex assay will reduce fluorescent signal crosstalk and background, but will also reduce the luminescence signal (about 1 order of magnitude). Also keep in mind that different brands of white plates will have varying degrees of auto-luminescence and crosstalk.

If your luminescence signal is too strong, you may want to switch to using a black solid-walled plate. The black plate absorbs some of the light of the reaction.

2. Avoid Bubbles

Try to avoid introducing bubbles into the well upon reagent addition. Bubbles in assay solutions cause light scattering and can result in erroneous signals when read on a luminometer.

3. Mix Your Samples Well

Mix your samples thoroughly for luminescent cell-based assays. Poor mixing can result in aggregation, precipitation, variations in reaction rates, or well-to-well concentration differences. Be sure to avoid creating droplets of reagent on the upper surfaces of the wells, because these can increase the likelihood of crosstalk.

4. Use Uniform Assay Volumes

Use the same volume across all wells: small differences in assay volume can cause large differences in signal. Use at least the minimum volume recommended by the instrument manufacturer.

5. Ensure Consistent Temperature

The temperature should be consistent across the plate. Luminescence assays are typically temperature-dependent, due to their enzymatic nature. While fluorescent assays may be incubated at room temperature or 37°C, luminescence assays should be incubated at the same temperature that the platereader is set to.

6. Time Reagent Incubation Carefully

The timing of reagent incubation for luminescent assays can be important. Reading the samples too soon may negatively impact the signal-to-noise ratio, sensitivity or dynamic range. Incubating too long may decrease the detection range by saturating the assay or detector with too much signal.

7. Don’t Compare Raw Signal Across Instruments

Instruments calculate relative light units (RLUs) by different methods, so it is important that you don’t compare the raw values across instruments, even if they are the same model. All instruments will have some level of background due to factors including the phosphorescence of plastics, differences in reagents, and electronic “noise”.

The dynamic range and sensitivity of an instrument is a key factor when measuring the extremes of very high or very low signal, and a sensitive instrument will have a clear distinction between the blank noise and the sample signal. We recommend comparing a positive control with a high signal to a background control in order to get a better understanding of the readings to expect for your instrument and experimental conditions.

8. No Wavelength Necessary

Don’t worry about selecting a wavelength for your assay—in most cases it isn’t necessary. Luminescence is a by-product of an enzymatic reaction, not light exciting fluorophores in the sample. This means that there is no need to filter out any light coming from a luciferase assay as you would with a fluorescence assay.

Bonus Tip: Let Your Instrument Do the Work For You

GloMax® microplate readers are already integrated with Promega assays and have the best sensitivity, broadest dynamic range and lowest crosstalk. Try one in your lab!

Still having problems? Our technical support scientists are here to help you with any issues you might have with a luminescent assay. In the meantime,

Keep calm and science on

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Julia worked as Science Writer at Promega before returning to UW-Madison to go back to the lab bench. She earned a B.S. in biology and a B.A. in chemistry from University of North Carolina Wilmington, and a PhD from University of Wisconsin-Madison. Her hobbies include reading fantasy novels, playing Magic: The Gathering, ultimate frisbee, Netflix, and long walks to the fridge.

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