Establishing luminescence-based assays: miniaturised experiments help to optimise results

Introduction

Today, luminescence-based assays are available for a broad range of applications, comprising e.g., gene reporter, cell viability and protein-protein interaction assays. These assays provide high sensitivity combined with a large dynamic range, and high-throughput compatibility on multiwell plate readers ^1-3. However, when establishing these assays users are often confronted with several complications. An effect, that especially affects luminescence measurements is cross-talk. 

Cross-talk paraphrases the effect, that a produced signal of a certain well is interfering with signals of its adjacent wells, resulting in artificially increased signals and reduced signal-to-blank values. Here, the cross-talk effect comprises the bleeding of a neighboring well’s signal into the optics from above the well as well as signal transmission through the well wall (fig. 1). 

In this AppNote we describe three different miniaturised experiments that enable users to identify and rapidly reduce cross-talk using the example of the BacTiter-Glo™ assay.

Assay Principle

The BacTiter-Glo™ assay is a straightforward "add, mix and measure” format which couples the available amount of ATP in a sample, a parameter proportional to the number of viable cells under optimal growth conditions, to the luciferase signal using the recombinantly modified firefly Ultra-Glo™ luciferase. Here the ATP is consumed to oxidatively decarboxylate luciferin to oxyluciferin resulting in an emission of yellow-green light with the emission maximum of ~550-560 nm (fig. 2). For more details on the reaction principle see (4).

(ii) To significantly reduce the signal bleeding of adjected wells, state-of-the-art microplate readers use apertures to physically isolate the emitted light of the target well (fig. 4A). In the case of the CLARIOstar Plus an aperture spoon is set up directly on top of the target well, letting its signal pass through a perforated screen into the optics while mostly excluding light from adjacent wells. Additionally, the CLARIOstar Plus offers a “cross-talk correction”, which mathematically corrects measured signals. Here, the signal bleeding and transmission of adjacent wells filled with blank samples were compared to blank wells situated next to wells containing a high ATP-concentration resulting in a factor dependent on the plate type (fig. 4B). Correction factors of 0.05353% for the white plates, 0.01277% for the grey plates and 0.00026% for the black plates were calculated. The increasing cross-talk effect from black via grey to white plates confirmed the results from the sensitivity measurements. 

(iii) Even when considering the above-mentioned effects on cross-talk and also applying the “correction factor” as well as the aperture spoon, the plate layout still decides if a measured signal is reproducible and correct. To elucidate this effect ATP standards S2, S4, S7 and blanks (fig. 3) were plated in a certain setup (fig. 5) and their measured signals were compared to the respective signals of the respective 7-point calibration (fig. 3).

The resulting relative deviation shows that the cross-talk effect decreases from white via grey to black plates and that cross-talk might also be relevant for wells not directly adjacent to each other. In addition, large dilution steps between adjected wells should be avoided, which is evident from the strong cross-talk between S7 and blank samples.