Quantifying Background Bioluminescence in C2C12 Cells Across Lumicycle Instruments

Abstract

Circadian rhythms are cell-autonomous mechanisms that drive repeating 24 hr cycles of gene expression and behavior. In circadian research, analysis of these rhythms can be tracked using self-luminous cells, such as those with the luciferase reporter coupled to a circadian clock gene (Bmal1). Real-time bioluminescence reporting technologies like the Lumicycle are equipped with photomultiplier tubes (PMTs), highly sensitive detectors of light in the green portion of the spectrum at which luciferase emits light. These detectors may detect nonrepresentative autofluorescence or background noise from different cell types and media sources. Therefore, this experiment tested consistency between machines to quantify the background luminescence signals. We collected 1 week of data from two Lumicycle 96 machines and analyzed rhythms with RStudio. These cell experiments produced comparable rhythms used to determine if accurate rhythmic activity was recorded. The development of a baseline correction protocol will allow valid comparison of cell experiments despite machine-dependent signal variability. Further optimization of software to reliably detect circadian rhythms plays an important role in further understanding circadian-related physiological disruption and disease.