Doug Wieczorek, Kyle Hooper and Michael Bjerke
June 2019; tpub_212
Crosstalk Between Wells
Crosstalk is a function of the opacity of the plates and the masking or isolation of one well from another by the detection instrument during reading. Luminescent studies are typically performed in opaque white plates for maximum luminescent output. Unfortunately, these white plates are not completely lightproof. A strong luminescent signal in one well will contribute light to an adjacent well with low or no signal (Figure 1). This is why you should never place a positive control (high luminescence) in a well next to a negative control (low or no luminescence).
A major factor in crosstalk is how well the luminescent reader isolates the signal from the well that is being measured from the signal coming from adjacent wells. Gaps between the well and the measuring device can let stray light into the detector. To circumvent this problem, you could design your experiment such that there are blank wells between your samples to avoid crosstalk from adjacent wells. However, this greatly reduces the number of samples you can measure on a microplate. If you have the option to simply use a luminometer with lower crosstalk, it’s best to avoid having to design your experiment around the performance of your plate reader.
Comparing Plate Readers
Sensitive assays also require a sensitive detection platform. Plate readers that are used to detect luminescence, fluorescence and absorbance signals can vary in sensitivity (Figure 2). Factors that contribute to an instrument’s sensitivity, or limit of detection, include background noise from the detector and electronics, the type of detector that is used, and the instrument’s configuration and overall design. Lowering the instrument’s background improves the limit of detection, which in turn improves the signal-to-noise ratio, providing more usable data from each experiment. Conversely, a high background can over shadow low‐level signals, reducing the usable data from each experiment. To get a true picture of the biology in the cell, it is more relevant to measure your assay at near physiological conditions if possible (Figure 3).
Figure 2. Sensitivity of luminescent detection on GloMax® systems and other commercially available plate readers.
The Bio-Glo™ Luciferase Assay System was used to test instrument sensitivity, and the limit of detection (LOD) was calculated. GloMax® Discover and Navigator had the lowest LOD at <10-19 moles luciferase.
To determine what amount of crosstalk you could expect from different commonly used microplate readers, we used the Kinase-Glo® Max Reagent (Cat. #V6071) and water to assemble plates where high luminescent wells were adjacent to no sample wells. The amount of signal detected in the wells of water was compared, and the results show that the amount of crosstalk varies dramatically between instruments (Table 1).
Table 1. Detection Instrument Comparison for Crosstalk between Wells.
High luminescent wells containing 100μl of a 100μM ATP solution plus 100μl of Kinase-Glo® Max Reagent were plated on a standard 96-well opaque white plate next to water-only wells (200µl Nuclease-Free Water). Signal in the water-only wells was measured after room temperature incubation for 10 min to achieve steady-state luminescence (luminescent half-life ~5 hours). A blank plate containing only 200μl of water/well was prepared to determine background. The background and experimental plates were read in the different microplate readers with 0.5sec integration times per well. The average background was determined for each instrument and this average was subtracted from the water-only and ATP/Kinase-Glo® Max wells. The average water-only RLUs were determined and the average ATP/Kinase-Glo® Max RLUs determined. The average water-only RLUs was divided by the average ATP/Kinase-Glo® Max RLUs to calculate the crosstalk value.