Microplate readers are essential tools for measuring biological responses and chemical reactions of samples in microplate wells.
Depending on your study, you can deduce various types of information, including cell viability, gene expression, bacterial growth, and concentrations of molecules such as DNA or proteins.
Poorly optimized assays cause misleading, inconsistent, or even non-existent results, leading to wasted time and resources.
In this guide to troubleshooting microplate assays, we highlight different microplate features and reader settings and the best strategies for obtaining high quality data.
The Value of Microplate Readers in Lab Research
The use of multi-mode microplate readers in research is so versatile that they feature in many scientific fields, such as:
- Drug discovery – Large libraries of compounds can be screened to aid drug discovery by measuring their effect on biological targets.
- Biotechnology – Gene expression and enzyme activity in multiple samples can be monitored.
- Disease research – Cellular and molecular anomalies can be identified, studied, and addressed.
- Environmental monitoring – Water samples can be tested for contaminants, such as bacteria, metals, or chemicals.
Translational benefits of microplate experiments include established assays such as ELISA tests to detect antigens; DNA, RNA, and protein quantification; and generating cell viability curves to monitor treatment response.
Despite the powerful benefits of microplate assays, the consequences of a poorly optimized experiment include:
- Misleading results, including false positives or false negatives, or inability to detect a signal at all;
- Longer read times than necessary;
- Inconsistent readings between experiments and within the microplate wells.
By using an appropriate experimental set-up and adjusting the settings on your microplate reader to suit your sample type, you can save valuable time, expensive reagents, and frustration.
Experimental Set-up
Choosing the Best Microplate Color. Which One is Right for Your Experiments?
The choice of microplate color can significantly impact the accuracy and efficiency of your readings, making it a critical first step in your experimental design.
Transparent
For absorbance assays, a typical clear polystyrene microplate is generally a good option to allow light through and limit background absorbance.
However, regular transparent microplates start to absorb light at wavelengths below 320 nm.
For assays that quantify DNA and RNA (A260), which require light transmission below 320 nm, cyclic olefin copolymer (COC) microplates are a better choice due to their transparency at these shorter wavelengths.
Black
Fluorescence intensity assays usually produce a strong signal.
The use of black microplates helps to reduce background noise and autofluorescence, providing better signal-to-blank ratios as the black plastic partially quenches the signal (Figure 1).
White
Luminescence assays often produce weak signals, even from concentrated samples.
White microplates are recommended to enhance these signals because they reflect the light from chemiluminescent reactions, effectively amplifying it (Figure 2).
Reducing Meniscus Formation
Absorbance measurements depend on the path length (depth) of your solution in the wells.
Longer path lengths increase absorbance values (Equation 1) because there are more molecules in the path of the incident light to absorb it.
The formation of a meniscus affects the path length and distorts absorbance measurements, leading to inaccurate concentration calculations.
A meniscus can also reflect emission light in fluorescence assays when top optics are used.
In designing your absorbance or fluorescence assay, there are several strategies you can take to reduce meniscus formation.
Use Hydrophobic Microplates
A meniscus is less likely to occur on a hydrophobic microplate.
While the typical polystyrene microplate is suitably hydrophobic, certain types that are optimized for cell culture, for example, become hydrophilic.
Treatments with hydroxylic and carboxylic groups increase the ability of the cells to adhere to the microplate surface but, in doing so, also increase the extent of the meniscus.
Where possible, opt for a hydrophobic microplate and avoid cell culture microplates for absorbance measurements.
Avoid Reagents Like TRIS, Acetate, and Detergents
Using compounds like Triton X, TRIS, EDTA, or sodium acetate can lead to increased meniscus formation as their concentrations rise (Figure 3). It’s advisable to minimize the use of these agents when possible.
Fill Wells to the Brim
Meniscus formation relies on having space along the edges of the wells for the solution to rise by capillary action. By calculating your solution volumes to fill the wells to their maximum capacity, you can minimize the space available for a meniscus to form.
Use a Path Length Correction Tool
The easiest option to account for the meniscus effect is to perform a path length correction, if the setting is available on your microplate reader.
This protocol detects the absorbance peak of water (970 nm) and uses this to determine the actual path length. The absorbance readings are then normalized to the fill volume.
Reducing Autofluorescence in Cell-based Assays
If you’re noticing significant background noise in your cell-based assay readings, it might be due to fluorescent molecules in your media supplements.
Fetal Bovine Serum and phenol red are common culprits because they contain aromatic side chains.
Consider using alternative media types, such as media optimized for microscopy or perform your measurements in phosphate-buffered saline with calcium and magnesium (PBS+).
Alternatively, you can set the reader to measure from below your microplate, circumventing the need for excitation and emission light to travel through the supernatant.
Optimizing Your Microplate Reader Settings
Sharpen Your Signal with the Right Gain Setting
The ‘gain’ is an artificial parameter applied to fluorescence- or luminescence-based readouts. It amplifies light signals making any variations more obvious and affecting the signal-to-background ratio.
Incorrectly selected gain settings can lead to saturation or poor-quality data.
The highest gain setting applies the highest possible signal amplification and allows the detector to separate low signals from blanks, making it appropriate for dim signals.
If your sample emits a bright signal, high amplification will cause the detector to oversaturate, resulting in unusable data. Lower gain settings are preferable in these cases.
If there is no automatic gain adjustment setting, you will need to adjust it manually. This can be done by measuring the highest signal in your plate, such as a positive control, to determine the maximum gain that can be applied without exceeding the signal range of the reader.
Check the Number of Flashes
If you are seeing an unusual amount of variability in your fluorescence or absorbance assays, you may need to check the ‘number of flashes’ setting.
Your microplate reader will direct light into a sample with either just one flash or several from which an average is taken.
A high number of flashes typically reduces variability and limits the background noise, as the average of different flashes levels out possible outliers, especially in samples with low concentrations.
However, beware that more flashes will extend the overall read time of your measurement, which could cause problems for kinetic experiments where you want short intervals between sample measurements.
In this case, you may want to select a lower number of flashes.
A balance is required to get accurate and reliable results without using excess time to make the measurement. For many assays 10–50 flashes will be sufficient.
Optimize Focal Height
If your signal intensity is much lower than expected, consider optimizing the focal height, i.e., the distance between the detection system and the microplate.
Signal intensity is usually highest slightly below the liquid surface of a sample, so it’s optimal to set the reading to occur at this point. In cell-based assays, where adherent cells reside at the bottom of the well, ensuring the focal height is adjusted to this layer can further improve accuracy and sensitivity.
Different models of microplate readers vary in their focal adjustment capabilities. Focal height may be:
- Fixed.
- Manually adjustable.
- Automatically determined.
If possible, use a well with a high expected signal for the focal adjustment. Some trial and error may be required to find the height with the highest signal intensity.
However, to get the best out of your assay, make sure all samples on your microplate have the same volume.
If you use the same focal height settings for subsequent measurements, make sure the volumes of the samples and type of microplate remain constant.
Check Your Well-Scanning Setting
Whether in fluorescence, luminescence, or absorbance assays, an uneven distribution of adherent cells, bacteria or precipitations could cause distorted readings.
If you are confused by distorted results, take a look at your well-scanning settings.
Instead of taking several measurements in the center of each well (a default setting in plate readers), well-scanning can be set to spread across the whole well surface in either an orbital or spiral scan pattern to correct for a heterogeneous signal distribution and obtain more reliable data (Figure 4).
The Benefits of BMG LABTECH Microplate Readers
A powerful microplate reader designed by specialists will help your experiments to run smoothly.
BMG LABTECH instruments are built to be extremely robust and reliable.
Due to their modularity, they can be equipped with different modes to cover a multitude of applications. This gives you the chance to build upon the complexities of your experiments as you go.
Adjust Gain Settings Throughout Your Assay
Signal intensity in kinetic assays typically builds up over time, increasing the risk of saturation—making an adjustment of the fluorescence gain upon detection complicated.
The Enhanced Dynamic Range (EDR) function on VANTAstar®, CLARIOstar® Plus, and PHERAstar® FSX readers eliminates the need for manual intervention.
This innovative EDR technology provides continuous and automatic gain adjustment during measurements and covers up to 8 decades of signal intensity, making gain adjustments superfluous.
Another benefit of EDR is that it allows for the comparison of data from different assay runs using the same assay kit at different times.
Although fluorescence intensity is not an absolute measurement, consistent comparisons are possible when the same assay conditions, microplate type, and detection settings are used.
Control Read Times with Flexible ‘Flash Number’ Settings
If you are using a high number of flashes, the time intervals between measurements can make a huge difference to your overall read time.
The BMG LABTECH software will automatically adjust it to the smallest possible interval to accommodate your flash number.
In addition, the total number of flashes can affect the longevity and, therefore, excitation intensity of your light source.
The Xenon flash lamps in BMG LABTECH microplate readers have a long lifetime, retaining 50 % of their emission intensity after 100,000,000 flashes.
Easily Pinpoint the Ideal Focal Height
BMG LABTECH readers have a focal height range of 0–25 mm when using the top optics, with the distance measured from the bottom of the microplate and a range of 0–9.7 mm for the bottom optics with the distance measured from the top to the bottom.
To help you find the optimal setting, the BMG LABTECH software automatically generates a focal height curve for you and selects the height with the highest signal intensity.
The helpful graph displays signal intensity across different focus levels (Figure 5). This visual tool makes it easier for you to pinpoint the ideal focal height for your measurements.
Account for Heterogeneity with Matrix Well-Scanning
You can go one step further than the orbital or spiral scanning options and acquire up to 900 measurement points across the whole well surface with the BMG LABTECH matrix scan feature.
Even with an uneven distribution of cells across a well, as can be the case with cell-based experiments and can potentially lead to a distortion of your data, you have the flexibility to exclude individual scan points or even entire sections of the well from your analysis.
To help you visualize your data, BMG LABTECH’s software offers a graphical display of each scan point. This feature creates a 3D heat map for each well, giving you a clear, comprehensive view of your results (Figure 6). This visual representation can be particularly useful in spotting trends or anomalies in your data.
Troubleshooting Microplate Assays Summarized
Whether you’ve discovered advanced instrument tools or are just setting out, optimizing your set-up is key to smooth microplate assay troubleshooting.
Adjusting the instrument’s focal height, gain, number of flashes, and well-scanning settings will significantly enhance the quality and reliability of your microplate assay data and save you time by avoiding common pitfalls.
Remember, the optimal instrument setting depends on the type of assay you are doing.
Troubleshooting is an ongoing process. If you believe you have tried everything to troubleshoot your microplate assay issues, consider contacting your equipment suppliers for additional support.
FAQs
Q: How can I choose the appropriate gain setting for my assay?
A: Start by setting the gain based on the intensity of your highest signal sample, like a positive control, to avoid saturation. Use the automatic gain adjustment if your microplate reader has this feature to optimize the signal-to-noise ratio throughout the assay.
For peace of mind and, for assays with varying signal intensities, consider using a reader with Enhanced Dynamic Range (EDR) technology to automatically adjust gain in real time.
Q: When should I use different colored microplates in my assays?
A: Choose clear microplates for absorbance assays where light transmission is crucial. Use black microplates to reduce background in fluorescence assays, enhancing signal clarity. White microplates are best for luminescence assays, as they reflect light and enhance signal intensity. Select the plate color based on your specific assay requirements to optimize data quality.
Q: What should I do if my absorbance readings are inconsistent?
A: Inconsistent absorbance readings can often be due to meniscus formation or incorrect path length. Use hydrophobic microplates, minimize agents like TRIS or detergents, and fill wells to the brim to reduce meniscus effects. Employ a path length correction protocol on your microplate reader for accurate normalization.
