Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.


Introduction
The use of luciferases as reporters of gene expression and protein activity has become a popular technique in molecular and cellular biology research. This is true in the circadian field, where the kinetics of firefly luciferase synthesis and catalytic inactivation are particularly well suited to reporting the longitudinal changes in gene expression that occur over the approximately 24 h circadian cycle. As such, luciferase is employed as a circadian reporter across a wide range of organisms, including fungi, plants, flies, and mammals 1,2,3,4 . When quantifying circadian gene expression in vitro, a photomultiplier tube (PMT) is commonly used to record the bioluminescent signal. PMTbased measurements have limited flexibility however, usually being restricted to a pre-determined plate or dish size. It is also not possible to collect any spatial information from samples monitored using a PMT, which can lead to a loss of information when imaging samples that show spatial variation in luciferase expression. Furthermore, as the PMT and associated electronics are prone to malfunction when exposed to the humidified environment of a standard cell culture incubator, longitudinal luciferase recording using PMTs is always performed in non-humidified incubators. In consequence, cell culture dishes must be sealed air-tight to prevent moisture loss through evaporation and the culture media must therefore be buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), rather than the CO 2 /bicarbonate buffering system that functions in vivo and is used routinely in mammalian tissue culture.

Seeding and Temperature Entrainment of Cells
NOTE: This protocol has been rigorously tested using primary and immortalized mouse fibroblasts expressing the PERIOD2::LUCIFERASE (PER2::LUC) fusion protein 4 . Adjustments may need to be made for experiments using other cell lines.
NOTE: Use of black sided, clear-bottomed plates is recommended as this allows culture health to be assessed before recordings begin whilst reducing interference between wells during recording. Where bioluminescence levels are extremely low, white dishes/plates should be used to maximize light detection, although please note that phosphorescence can lead to increased background signal that persists for several hours. 9. Return the plates to the incubator and allow monolayers to reach 100% confluence (approximately 7 days).
NOTE: Once confluent, cell lines that inhibit contact can be maintained for up to three weeks prior to experimentation provided that the media is refreshed regularly (every 5-7 days). 10. Once confluent, synchronize cellular rhythms using applied temperature cycles (12 h at 32 °C, 12 h at 37 °C) for a minimum of 72 h immediately prior to experimentation 5,6 (using a pre-programmed cycling incubator, controlled by a computer connected to the incubator through a serial RS-232 port). NOTE: The number of temperature cycles required for synchronization of cellular rhythms may vary between cells lines and may therefore need to be optimized for other cell lines. Acute pharmacological stimulation using forskolin or dexamethasone has also been used previously to synchronize cellular rhythms 7,8 .

NS21 Preparation
NOTE: NS21 is a serum-free supplement for the maintenance of neuronal and other cell cultures. It is a refinement of a similar supplement known as B27, which is commercially available and can be used as a serum replacement during circadian bioluminescence recordings 9 . Either supplement can be used interchangeably in the recording media for the experimental protocols described below. It is quite feasible and cost effective to make NS21 in-house, as in Chen et al. 10 , and as described here.
1. Equilibrate all components described in Table 1 at room temperature for 1 h before beginning. 2. Dissolve 50 g bovine albumin in 324 mL basal medium (e.g., neurobasal) on ice. Stir the mixture as little as possible. 3. Add all other components, stirring minimally after each component but still ensuring thorough mixing. Aim to dissolve all components within 90 min of starting. 4. Aliquot the final mixture and store at -20 °C until required. Avoid repeated freeze-thaw cycles.
NOTE: The mixture is too viscous to filter at this stage, but will be sterile filtered when diluted with media before adding it to cells.

Recording Media Preparation
NOTE: A primary advantage of the bioluminescence incubator over other equipment for recording longitudinal bioluminescence is that, by virtue of being able to humidify the incubator and vary the partial pressures of O 2 and CO 2 , it is possible to use a wider range of media conditions for recording bioluminescence -including conditions which more closely approximate the physiological niche occupied by different cell types in vivo. Below we describe the formulation of recording media adapted from Hastings et al. 9 , that we have used routinely with cultured fibroblasts and other cell types. The first is for sealed culture conditions (without gas exchange), and the second is for physiologically relevant conditions and should be used under humidified conditions with 5% CO 2 . Many other variations are both possible and advisable, depending on the exact application and cell type.  Figure 1A shows an example video of a bioluminescence recording from 6 x 96-well plates containing PER2::LUC fibroblasts 4 . The outermost wells do not contain cells as these were not required for this experiment. Cells have undergone differential temperature entrainment, whereby they undergo either temperature cycle of 12 h, at 32 °C followed by 12 h 37 °C for 72 h or the converse (12 h, at 37 °C followed by 12 h, at 32 °C for 72 h), before being held at constant 37 °C for recording. 60 min exposures were taken every hour. Two of these conditions are quantified in Figure 1B. Figure 2A shows a schematic of the setup of a system for perfused tissue culture. This consists of two channel slides connected with tubing. Media is driven across the cells by a syringe pump. The first of these slides acts as a gas permeable bubble trap (buffer slide) and contains no cells, with the second containing the cells from which bioluminescence is recorded. A representative recording video from this system is shown in Figure 2B. 15 min exposures were taken every 15 min. Here, cells are maintained either under standard perfusion conditions or in the presence of casein kinase inhibitor PF670462, which has been previously shown to lengthen circadian period and reduce the amplitude of clock gene expression rhythms in cultured mammalian cells 14 . The effect on PER2::LUC expression is shown in Figure 2C (top panel) against cells treated with the same concentration of drug under standard static cell culture conditions shown in Figure 2C (bottom panel), with quantification of period shown in Figure 2D. It is clear from this that treatment with PF670462 influences PER2::LUC expression under both sets of conditions. However, whilst cells treated with PF670462 under perfused conditions show period lengthening of approximately 3 h (3 ±0.9 h), cells in static conditions treated with the drug show substantially greater period lengthening to a period of >48 h. This can be fit by a damped cosine, as described in Section 7, (extra-sum-of-squares F test versus a straight line, p <0.0001). Interestingly, the magnitude of period lengthening under perfusion is much closer to that observed in vivo

Discussion
The protocol described here is for mammalian cell culture, both under perfused and static conditions. However, the ALLIGATOR can easily be adapted to other model systems. Indeed, it has already been shown to provide an excellent platform for simultaneous monitoring of locomotion, sleep, and peripheral gene expression rhythms in Drosophila melanogaster maintained under constant darkness 15 . It is also noted that, depending on the application, camera types other than that mentioned here may be appropriate. We envisage that with the appropriate filters, a modified version of the current setup could in principal be used for fluorescence quantification.
The only applications for which the ALLIGATOR might not be appropriate are those for which particularly high spatial resolution is required, such as imaging of spatiotemporal organization of PER2::LUC expression in organotypic slices of the mammalian suprachiasmatic nucleus, or other small tissue slices.
The ALLIGATOR enables many experiments to be performed that hitherto have not been readily achievable by conventional recording techniques. Compared with current methods for the measurement of bioluminescence, the ALLIGATOR provides increased flexibility in both the type of cell culture dish or slide that can be used, the external media conditions, sensitivity, and processivity. This is particularly relevant at a time when there is a move away from standard 2D cell culture models towards 3D organoid and flow culture systems. As such, it is anticipated that the ALLIGATOR will provide an adaptable method by which bioluminescence can be measured over many days and weeks under a broad range of conditions.