We would like to thank Cairn Research for working with us to develop this system, in particular Mark Henson, Jeremy Graham and Joao Correia. We also thank David Welsh and Akhilesh Reddy for valuable discussion during the design phase, as well as Peter Laskey (formerly of Hamamatsu) for arranging the loan of a demo camera and David Wong for his critical input to the manuscript.

1. 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 protein4. Adjustments may need to be made for experiments using other cell lines.
<ol>
	<li>Prior to beginning cell culture, place the following into a 37 &#176;C water bath to warm: phosphate-buffered saline (PBS) (pH 7.4), PBS supplemented with 0.68 mM ethylenediaminetetraacetic acid, pH 7.2 (PBS+EDTA), appropriate cell culture media, e.g., Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 &#181;g/mL streptomycin, and 0.25% trypsin solution.</li>
	<li>Dilute trypsin 1:3 with pre-warmed PBS+EDTA solution and return to the water bath.</li>
	<li>Take a 125 cm2 flask of luciferase-expressing cells that are 70-90% confluent. Aspirate the media. Add 10 mL pre-warmed PBS.</li>
	<li>Aspirate the PBS. Add 2.5 mL pre-warmed trypsin-EDTA and return the flask to a 37 &#176;C incubator for 5 min.</li>
	<li>Remove the cells from the incubator and view under a microscope. Once the majority of cells have detached from the bottom of the flask, continue to the next step. If most cells remain attached to the bottom of the flask, return it to the incubator until most cells are in suspension.</li>
	<li>Add a further 7.5 mL of standard cell culture medium to the flask. Optionally, remove 1 mL of this for further passage of the cells, as required.</li>
	<li>Count the density of the remaining cells using a hemocytometer and trypan blue. Dilute the cells further, as appropriate for that cell line. 
	NOTE: For the experiments below, PERIOD2::LUC fibroblasts were seeded between 6 x 103 cells/cm2 and 1 x 104 cells/cm2 in either 96-well plates, 35 mm dishes, or channel slides.</li>
	<li>Seed cells onto the tissue culture dishes or plates that will be used for recording. 
	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.</li>
	<li>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).</li>
	<li>Once confluent, synchronize cellular rhythms using applied temperature cycles (12 h at 32 &#176;C, 12 h at 37 &#176;C) for a minimum of 72 h immediately prior to experimentation5,6 (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 rhythms7,8.</li>
</ol>
2. 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 recordings9. 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.
<ol>
	<li>Equilibrate all components described in Table 1 at room temperature for 1 h before beginning.</li>
	<li>Dissolve 50 g bovine albumin in 324 mL basal medium (e.g., neurobasal) on ice. Stir the mixture as little as possible.</li>
	<li>Add all other components, stirring minimally after each component but still ensuring thorough mixing. Aim to dissolve all components within 90 min of starting.</li>
	<li>Aliquot the final mixture and store at -20 &#176;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.</li>
</ol>
3. 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 O2 and CO2, it is possible to use a wider range of media conditions for recording bioluminescence &#8212; 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% CO2. Many other variations are both possible and advisable, depending on the exact application and cell type.
<ol>
	<li>MOPS-buffered high glucose media preparation 
	NOTE: Stock solution (1 L):DMEM powder (8.3 g/L), 0.35 mg/mL sodium bicarbonate, 5 mg/mL glucose, 0.02 M MOPS, 100 U/mL penicillin, 100 &#181;g/mL streptomycin.
	<ol>
		<li>Dissolve 8.3 g DMEM powder in 900 mL ultrapure water in a 1,000 mL measuring cylinder and stir for 30 min until all particles are dissolved.</li>
		<li>Add 50 mL of glucose solution (100 g/L), 20 mL of MOPS (1 M, pH 7.6), 10 mL of 100x pen/strep solution, and stir for a further 10 min.</li>
		<li>Add 4.7 mL 7.5% sodium bicarbonate solution and stir for a further 5 min.</li>
		<li>Adjust the media pH to 7.6 (if at room temperature) or 7.4 (37 &#176;C) with HCl/NaOH.</li>
		<li>Add ultrapure water to produce a final volume of 1,000 mL.</li>
		<li>Sterilize by filtration through a 0.22 &#181;m filter and store at 4 &#176;C until required.</li>
		<li>Prior to experimentation, supplement an appropriate volume of stock solution with 10% serum, 2mM L-alanyl-L-glutamine, 2% NS21, and 1 mM luciferin to make a working stock. Pass this stock through a 0.22 &#181;m sterile filter.</li>
		<li>Measure osmolality of the media using an osmometer.
		<ol>
			<li>Turn on the osmometer.</li>
			<li>Calibrate the osmometer, first with ultrapure water. Add 50 &#181;L water to the bottom of a measuring vessel. Ensure there are no bubbles in the liquid. Clip the vessel over the probe. Press &#39;Zero&#39; and carefully lower the osmometer arm to the lowest point. Wait until reading is complete and green &#39;result&#39; light is lit before raising the arm and removing the vessel.</li>
			<li>Repeat to calibrate the osmometer to 300 mOsmol/kg using 50 &#181;L calibration standard. Press &#39;Cal&#39; before lowering the arm.</li>
			<li>Measure the sample osmolality by adding 50 &#181;L of media to the bottom of a measuring vessel and measure as above. Press &#39;sample&#39; before lowering the osmometer arm.</li>
		</ol>
		</li>
		<li>Adjust the osmolality to 350 mOsmol using 5 M NaCl.</li>
	</ol>
	</li>
	<li>HCO3-buffered low glucose media (perfusion medium) preparation 
	NOTE: Stock solution (1 L): DMEM powder (8.3 g/L), 3.7 mg/mL sodium bicarbonate, 1 mg/mL glucose, 100 U/mL penicillin, 100 &#181;g/mL streptomycin.
	<ol>
		<li>Dissolve 8.3 g DMEM powder in 900 mL ultrapure water in a 1,000 mL measuring cylinder and stir for 30 min until all particles are dissolved.</li>
		<li>Add 50 mL of glucose solution (100 g/L), 10 mL of 100x pen/strep solution and stir for a further 10 min.</li>
		<li>Add 49.4 mL 7.5% sodium bicarbonate solution and stir for a further 5 min.</li>
		<li>Adjust the media pH to 7.6 (at room temperature) or 7.4 (37 &#176;C) with HCl/NaOH.</li>
		<li>Add ultrapure water to give a final volume of 1,000 mL.</li>
		<li>Pass the solution through a 0.22 &#181;m sterile filter and store at 4 &#176;C until use.</li>
		<li>Prior to experimentation, supplement an appropriate volume of stock solution with 2% serum, 2 mM L-alanyl-L-glutamine, 2% NS21 and 1 mM luciferin to make a working stock. Pass this stock through a 0.22 &#181;m sterile filter.</li>
		<li>Measure and adjust the osmolality to 350 mOsmol using 5 M NaCl, as for the MOPS-buffered media.</li>
	</ol>
	</li>
</ol>
NOTE: Luciferin concentration should be determined empirically for each cell type and context. For further information see Feeney et al.11 Serum and NS21 (or B27 if used) concentrations can be varied depending on application. However, we do advise that, unless empirically tested to show otherwise, serum and NS21 (or alternative serum-free supplement) be used, as these promote cell survival and attachment. In cell lines that do not contact inhibit well, it may be necessary to lower the serum concentration in the recording media to prevent confounding effects of proliferation, which is also promoted by serum.
4. Recording
<ol>
	<li>Prior to beginning the recording, place the appropriate working media stock (from step 3.1 or 3.2 depending on the experiment) in a 37 &#176;C water bath to warm.</li>
	<li>Whilst the media is warming, focus the camera in the bioluminescence incubator.
	<ol>
		<li>Unscrew the water-impermeable, heated neutral density filter and set aside.</li>
		<li>Set the software to record a video with a high acquisition rate. Click &#34;Acquisition | Acquisition Setup&#34;. Set &#39;Exposure time&#39; to 0.2 s and kinetic cycle time to 0.3 seconds. Make sure that EM gain is turned off.</li>
		<li>Close the &#39;Acquisition Setup&#39; window and click the &#34;take video&#34; icon.</li>
		<li>Using the focusing cylinder, rotate the camera lens until items on the shelf at the height to be recorded are clearly in focus in the video.</li>
		<li>Stop the video by clicking the &#39;stop video&#39; icon.</li>
		<li>Screw the heated neutral density filter back into position; failure to do so will result in damage to the EM-CCD camera and/or fogging of the objective due to condensation.</li>
	</ol>
	</li>
	<li>Set O2, CO2, and temperature to the desired experimental conditions using the control panel on the front of the bioluminescence incubator. 
	NOTE: If performing perfused cell culture proceed directly to Section 5 at this stage.</li>
	<li>Remove the cells from the tissue culture incubator and aspirate off the media. Replace the media with pre-warmed working stock.</li>
	<li>If the bioluminescence incubator is not to be humidified during the experiment, then seal the dishes and plates with a gas-impermeable seal. If the bioluminescence incubator is to be humidified, then it is not strictly necessary to seal dishes, although it is preferable to seal dishes with a small volume, such as 96-well plates, using a gas-permeable seal, as a small quantity of evaporation can otherwise still occur even in humidified incubators. 
	NOTE: Humidification is achieved by filling the water tray at the base of the incubator.</li>
	<li>Transfer the cells to the bioluminescence incubator, close the incubator door and secure incubator cover in place to form a light-tight seal.</li>
	<li>Select the recording parameters appropriate for the application.
	<ol>
		<li>Click &#34;Acquisition| Acquisition Setup&#34;.</li>
		<li>In the &#39;Camera Setup&#39; panel, set: &#39;Acquisition mode&#39; to &#39;Kinetic&#39;; &#39;Exposure time&#39; to desired exposure time, in seconds (see note below); &#39;Accumulations&#39; to 1; &#39;Kinetic Series length&#39; to number of acquisition cycles desired; &#39;Kinetic cycle time&#39; to desired imaging interval (ensure that this is greater than the exposure time); &#39;Shift speed&#39; to 4.33 &#181;secs; &#39;Gain&#39; to 1; and &#39;EM gain&#39; to the desired level (see note below)</li>
		<li>In the &#39;Autosave&#39; panel, set the file-type to .sif or .tif. Provide a file name and appropriate saving location.</li>
		<li>In the &#39;Spooling&#39; panel, set the file-type to tiff. Provide a file name and appropriate saving location. Close the acquisition setup window.</li>
	</ol>
	</li>
	<li>Start recording by clicking the &#39;take signal&#39; button. 
	NOTE: As the bioluminescence incubator can be used for a large number of different cell and tissue types, all of which will have different levels of bioluminescence, the exposure time required to collect an appropriate bioluminescent signal will vary between experiments. It is also possible to vary the electron-multiplying (EM) gain of imaging in order to increase the magnitude of the signal collected. For new applications of unknown brightness, we suggest first taking a single image with a relatively short exposure time (around 10 min) without EM gain and subsequently adjusting both exposure time and EM gain until the desired recording conditions have been reached. In most cases, a mean signal of 10-20% of the maximum possible pixel intensity for the camera is a good level to aim for. Once an appropriate set of recording parameters has been reached, start longitudinal acquisition of images, as described above.</li>
</ol>
5. Perfused Tissue Culture (Optional)
NOTE: As described in the introduction, the bioluminescence incubator is suitable for imaging of non-standard tissue culture systems. This is exemplified in the development of a system for perfused cell culture.
<ol>
	<li>Seed the cells onto single channel slides with Luer connectors with a channel depth of 0.6 or 0.8 mm, in DMEM with 10% FBS and pen/strep, as described in Section 1. Make the perfusion media as described in step 3.2.</li>
	<li>Prior to recording, prepare tubing for the required perfusion system using 1 mm internal diameter (I.D.) ETFE tubing and 1 mm I.D. silicone tubing, elbow, male and female Luer fittings, as shown in Figure 2A. 
	NOTE: The majority of the length of the tubing is ETFE tubing, with 2 cm sections of silicone tubing used to connect the ETFE tubing to the Luer fittings, which subsequently link into the channel slides.</li>
	<li>Sterilize the tubing by flushing with 70% ethanol, followed by sterile PBS.</li>
	<li>Remove the cell cultures in channel slides from the incubator. Aspirate the media and replace with pre-warmed perfusion media.</li>
	<li>Fill a 20 mL syringe with the pre-warmed perfusion media.</li>
	<li>Connect the tubing to the buffer slide (see Figure 2) but not to the cell-containing slide, and flush the tubing and slide with perfusion media (3-5 mL).</li>
	<li>Connect the cell-containing slide and flush the whole system with a further 1 mL of media to remove any air bubbles.</li>
	<li>Transfer the entire system to the bioluminescence incubator.</li>
	<li>Fit the media-filled syringes to the syringe pump without disconnecting from the tubing and flush a further 1 mL of media through the entire system using the pump to ensure there are no air bubbles in the system.</li>
	<li>Set the diameter on the pump to that of the syringe being used. 
	NOTE: For the 20 mL syringes used here, the diameter is 19.13 mm.</li>
	<li>Set the flow rate of the pump to 50 &#181;L/h and start it running.</li>
	<li>Ensure that there are no bubbles in any slides or tubing before the start of recording, as these will influence luciferase expression when they pass across the cell monolayer. If necessary, flush further media across the cells to achieve this.</li>
	<li>Close the bioluminescence incubator door and continue as in step 4.7 to begin recording.</li>
</ol>
6. Treatment During Recording
NOTE: Sometimes it is desirable to treat cells midway through a recording, be it with pharmacological or hormonal agents. In such cases, it is imperative that the cells are handled with care to prevent the cellular oscillation from resetting during treatment. For this reason, it is of particular importance that the cells are maintained at a constant temperature, as this is a major entraining cue for cellular circadian rhythms5,6.
<ol>
	<li>Prepare all components of treatment prior to stopping recording.</li>
	<li>Prepare a chemical isothermal pad at 37 &#176;C.</li>
	<li>Stop the device recording, remove the dishes to be treated and place on the isothermal heat pad.</li>
	<li>Treat the cultures as required and return to the bioluminescence incubator in the same location as before.</li>
	<li>Re-start recording.</li>
</ol>
7. Analysis
NOTE: The bioluminescence incubator produces data in the form of a series of individual images. We primarily use Fiji12 to manage these images and then export the mean pixel intensity data for each region-of-interest (ROI) for further analysis.
<ol>
	<li>Open the image stack in Fiji and adjust the brightness and contrast by clicking &#34;Image| Adjust| Brightness/Contrast&#34; and adjusting the sliding bars until images are within an appropriate range for viewing the&#160; bioluminescent signal.</li>
	<li>Select areas of interest using the ROI manager available under &#34;Analyze| Tools| ROI Manager&#34;.</li>
	<li>Export mean signal for the selected area by clicking &#34;ROI manager| More| Multimeasure&#34;.</li>
	<li>Copy the resulting data into analysis software.</li>
	<li>Manually adjust the time base of data to the time interval of imaging, (i.e., 15, 30 or 60 min intervals, described as 0.25, 0.5, or 1 h) rather than the image number provided by Fiji. 
	NOTE: Further analysis can now be performed, such as determination of period. For this, the following equation is used to perform non-linear regression analysis: 
	<img alt="Equation" src="/files/ftp_upload/56623/56623eq1.jpg" /> 
	where y is the signal, x the corresponding time, m is the gradient of the trend line, c is the y intercept of the trend line, Amplitude is the height of the peak of the waveform above the trend line, k is the decay constant or rate of damping (such that 1/k is the half-life), phase is the shift in x of the cosine wave, and the period is the time taken for a complete cycle to occur13. R2 value is used as an indicator of goodness-of-fit.&#160; Other analysis methods are also possible.&#160;The first 24 h of the acquisition should be excluded from analysis, as cellular bioluminesence may exhibit transient, non-circadian changes during this time. This is the result of an acute response to media change.</li>
</ol>

<ol>
	<li>Morgan, L. W., Greene, A. V., Bell-Pedersen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+and+light-induced+expression+of+luciferase+in+Neurospora+crassa.">Circadian and light-induced expression of luciferase in Neurospora crassa.</a> Fungal Genet. and Biol. 38 (3), 327-332 (2003).</li><li>Millar, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Circadian+Phenotype+Based+on+Firefly+Luciferase+Expression+in+Transgenic+Plants.">A Novel Circadian Phenotype Based on Firefly Luciferase Expression in Transgenic Plants.</a> Plant Cell Online. 4 (9), 1075-1087 (1992).</li><li>Yu, W., Hardin, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Firefly+Luciferase+Activity+Assays+to+Monitor+Circadian+Molecular+Rhythms+In+Vivo+and+In+Vitro.">Use of Firefly Luciferase Activity Assays to Monitor Circadian Molecular Rhythms In Vivo and In Vitro.</a> Circadian Rhythms: Methods and Protocols. , 465-480 (2007).</li><li>Yoo, S. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PERIOD2::LUCIFERASE+real-time+reporting+of+circadian+dynamics+reveals+persistent+circadian+oscillations+in+mouse+peripheral+tissues.">PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues.</a> Proc Natl Acad Sci. 101 (15), 5339-5346 (2004).</li><li>Buhr, D. E., Yoo, S. -. H., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+as+a+universal+resetting+cue+for+mammalian+circadian+oscillators.">Temperature as a universal resetting cue for mammalian circadian oscillators.</a> Science. 330 (6002), 379-385 (2010).</li><li>Brown, S. A., Zumbrunn, G., Fleury-Olela, F., Preitner, N., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythms+of+mammalian+body+temperature+can+sustain+peripheral+circadian+clocks.">Rhythms of mammalian body temperature can sustain peripheral circadian clocks.</a> Current Biology. 12 (18), 1574-1583 (2002).</li><li>Balsalobre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resetting+of+circadian+time+in+peripheral+tissues+by+glucocorticoid+signaling.">Resetting of circadian time in peripheral tissues by glucocorticoid signaling.</a> Science. 289 (5488), 2344-2347 (2000).</li><li>Balsalobre, A., Marcacci, L., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+signaling+pathways+elicit+circadian+gene+expression+in+cultured+Rat-1+fibroblasts.">Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts.</a> Curr. Biol. CB. 10 (20), 1291-1294 (2000).</li><li>Hastings, M. H., Reddy, A. B., McMahon, D. G., Maywood, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circadian+mechanisms+in+the+suprachiasmatic+nucleus+by+transgenesis+and+biolistic+transfection.">Analysis of circadian mechanisms in the suprachiasmatic nucleus by transgenesis and biolistic transfection.</a> Methods Enzymol. 393, 579-592 (2005).</li><li>Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Ba Barres, ., Hell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NS21:+re-defined+and+modified+supplement+B27+for+neuronal+cultures.">NS21: re-defined and modified supplement B27 for neuronal cultures.</a> J. Neurosci Methods. 171 (2), 239-247 (2008).</li><li>Feeney, K. A., Putker, M., Brancaccio, M., ONeill, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+Characterization+of+Firefly+Luciferase+as+a+Reporter+of+Circadian+Gene+Expression+in+Mammalian+Cells.">In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.</a> J. Biol. Rhythms. 31 (6), 540-550 (2016).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Hirota, T., Lewis, W. G., Liu, A. C., Lee, J. W., Schultz, P. G., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+biology+approach+reveals+period+shortening+of+the+mammalian+circadian+clock+by+specific+inhibition+of+GSK-3beta.">A chemical biology approach reveals period shortening of the mammalian circadian clock by specific inhibition of GSK-3beta.</a> Proc Natl Acad Sci. 105 (52), 20746-20751 (2008).</li><li>Meng, Q., Maywood, E. S., Bechtold, D. A., Lu, W., Li, J., Gibbs, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrainment+of+disrupted+circadian+behavior+through+inhibition+of+casein+kinase+1+(+CK1+)+enzymes.">Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 ( CK1 ) enzymes.</a> Proc Natl Acad Sci. 1, 1-6 (2010).</li><li>Khabirova, E., Chen, K. -. F., O'Neill, J. S., Crowther, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flyglow:+Single-fly+observations+of+simultaneous+molecular+and+behavioural+circadian+oscillations+in+controls+and+an+Alzheimer's+model.">Flyglow: Single-fly observations of simultaneous molecular and behavioural circadian oscillations in controls and an Alzheimer's model.</a> Sci. Rep. 6, 33759 (2016).</li></ol>

No conflicts of interest exist.

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 darkness15. 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.

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 mammals1,2,3,4.
When quantifying circadian gene expression in vitro, a photomultiplier tube (PMT) is commonly used to record the bioluminescent signal. PMT-based 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 CO2/bicarbonate buffering system that functions in vivo and is used routinely in mammalian tissue culture.
As a result of these limitations, measurement of bioluminescence by PMTs usually places tight restrictions upon the conditions under which cells are maintained during experiments. To overcome these problems, and also to increase the range of possible experimental conditions, we use a standard CO2/N2 170 L tissue culture incubator that has been adapted by the addition of a water-chilled electron-multiplying charge-coupled device (EMCCD) camera with anti-mist optics and digital control of temperature and gas levels. This has been dubbed an Automated Longitudinal Luciferase Imaging Gas and Temperature-Optimized Recorder, or ALLIGATOR. The ALLIGATOR allows for substantially increased flexibility of bioluminescent imaging, both for high-throughput imaging of standard tissue culture plates (up to 6 x 96- or 384-well plates simultaneously) and also for non-standard tissue culture systems, such as perfused cells grown in microfluidic devices. This instrument also allows for imaging to occur under humidified conditions and with variable control of both CO2 and O2 partial pressure as well as temperature.
The protocol below describes a method for the bioluminescent recording of mammalian cell and tissue culture systems using an ALLIGATOR (henceforth referred to as &#39;bioluminescence incubator&#39;). It should be noted, however, that the system would be well suited to bioluminescent imaging and also, with some modification, fluorescent imaging, in a number of other biological systems and contexts.

<table><tbody><tr><td>DMEM (1x) + GlutaMAX</td><td>Gibco</td><td>31966-021</td><td></td></tr><tr><td>Hyclone FetalClone III Serum</td><td>GE Healthcare</td><td>SH30109.03</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermofisher</td><td>21103049</td><td>basal medium</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A4919</td><td></td></tr><tr><td>Catalase</td><td>Sigma</td><td>C40</td><td></td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G6013</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I1882</td><td></td></tr><tr><td>Superoxide Dismutase</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>Holo-transferrin</td><td>Calbiochem</td><td>616424</td><td></td></tr><tr><td>T3 (triiodo-L-thyronine</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>L-Carnitine</td><td>Sigma</td><td>C7518</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma</td><td>E9508</td><td></td></tr><tr><td>D (+)-Galactose</td><td>Sigma</td><td>G0625</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Sodium Selenite</td><td>Sigma</td><td>S9133</td><td></td></tr><tr><td>Corticosterone</td><td>Sigma</td><td>C2505</td><td></td></tr><tr><td>Linoleic Acid</td><td>Sigma</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma</td><td>L2376</td><td></td></tr><tr><td>Lipoic Acid</td><td>Sigma</td><td>T1395</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Retinol Acetate</td><td>Sigma</td><td>R7882</td><td></td></tr><tr><td>Retinol, all trans</td><td>Sigma</td><td>95144</td><td></td></tr><tr><td>D,L-alpha-Tocopherol</td><td>Sigma</td><td>95240</td><td></td></tr><tr><td>D,L-alpha-Tocopherol acetate</td><td>Sigma</td><td>T3001</td><td></td></tr><tr><td>Sodium Bicarbonate Solution</td><td>Sigma</td><td>S8761-100ML</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Galaxy 170R incubator&amp;nbsp;</td><td>Eppendorf</td><td>CO17301001</td><td></td></tr><tr><td>Luciferin</td><td>Biosynth</td><td>L-8220</td><td></td></tr><tr><td>D -(+)-Glucose solution</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>DMEM powder</td><td>Sigma</td><td>D5030</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>PHG0007</td><td></td></tr><tr><td>1 mm I.D. silicone tubing&amp;nbsp;</td><td>GE Healthcare</td><td>19-4692-01</td><td></td></tr><tr><td>Elbow luer connector</td><td>Ibidi</td><td>10802</td><td></td></tr><tr><td>Male luer fittings</td><td>Ibidi</td><td>10826</td><td></td></tr><tr><td>Female luer fittings</td><td>Ibidi</td><td>10825</td><td></td></tr><tr><td>&amp;micro;-slide luer I 0.6</td><td>Ibidi</td><td>80196</td><td></td></tr><tr><td>BD plastipak 20ml syringe</td><td>Becton Dickinson</td><td>300613</td><td></td></tr><tr><td>1mm I.D. ETFE tubing</td><td>GE Healthcare</td><td>18-1142-38</td><td></td></tr><tr><td>PF670462</td><td>Sigma</td><td>SML0795</td><td></td></tr><tr><td>B27 Supplement (50x)</td><td>ThermoFisher</td><td>17504044</td><td></td></tr><tr><td>iXon Ultra EMCCD camera</td><td>Andor</td><td>iXon 888</td><td></td></tr><tr><td>Fiji</td><td>ImageJ</td><td>N/A</td><td></td></tr><tr><td>Prism 7.0</td><td>Graphpad Software</td><td>N/A</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Deltaphase Isothermal Pad</td><td>Braintree Scientific</td><td>39DP</td><td></td></tr><tr><td>Heated neutral density filter&amp;nbsp;</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>Osmomat 030</td><td>Gonotech</td><td>Discontinued</td><td></td></tr><tr><td>300 mOsmol/kg calibration standard</td><td>Gonotech</td><td>30.9.0020</td><td></td></tr><tr><td>Measuring vessel</td><td>Gonotech</td><td>30.9.0010</td><td></td></tr><tr><td>Focusing cylinder</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>NE-1600 programmable syringe pump</td><td>Pump Systems inc.</td><td>NE-1600</td><td></td></tr><tr><td>Andor Solis Software</td><td>Andor</td><td>N/A</td><td></td></tr></tbody></table>

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.

This article outlines a protocol for the bioluminescent imaging of mammalian cells using an ALLIGATOR (bioluminescence incubator). This technique allows for flexibility of physical setup and extracellular conditions when imaging bioluminescent systems. Methods for both simple static tissue culture (Figure 1, Supplementary Video 1) and perfused cell culture (Figure 2, Supplementary Video 2) are described, but many other setups could be imaged using this system. All data were quantified using the methods described in Section 7.
Figure 1A shows an example video of a bioluminescence recording from 6 x 96-well plates containing PER2::LUC fibroblasts4. 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 &#176;C followed by 12 h 37 &#176;C for 72 h or the converse (12 h, at 37 &#176;C followed by 12 h, at 32 &#176;C for 72 h), before being held at constant 37 &#176;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 cells14. 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 &#177;0.9 h), cells in static conditions treated with the drug show substantially greater period lengthening to a period of &#62;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 &#60;0.0001). Interestingly, the magnitude of period lengthening under perfusion is much closer to that observed in vivo14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56623/56623fig1v2.jpg" /> 
Figure 1: Data example. (A) Example video snapshot of bioluminescence from immortalized PER2::LUC in 6 x 96 well plates in the bioluminescence incubator. Wells to be quantified have been highlighted in yellow in the Supplementary Video 1. (B) Quantification of bioluminescence from A. Two sets of 3 plates of cells were entrained using temperature cycles (12 h, at 32 &#176;C; 12 h, at 37&#176;C) that were anti-phasic to each other to produce opposite phases of PER2 protein expression (n = 6, mean &#177; SEM). <a href="https://cloudflare.jove.com/files/ftp_upload/56623/56623fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56623/56623fig2v2.jpg" /> 
Figure 2: Perfusion with CK1&#948; inhibitors. (A) Schematic of perfusion system. (B) Example video snapshot of a bioluminescence recording from PER2::LUC cells under perfusion. A sample area of quantification is highlighted in yellow. (C) Quantification of bioluminescence of PER2::LUC fibroblasts under perfusion and in static conditions with and without 3 &#181;M CK1 inhibitor PF670462 (n = 3, mean &#177; SEM). Bioluminescence has been normalized to minimum and maximum values. (D) Analysis of period (Two-way ANOVA, Holm-Sidak&#39;s multiple comparisons test). <a href="https://cloudflare.jove.com/files/ftp_upload/56623/56623fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Final Medium Concentration (&#956;M)</td>
			<td>Stock (mg/mL)</td>
			<td>For 400 mL NS21</td>
		</tr>
		<tr>
			<td>Bovine Albumin</td>
			<td>37</td>
			<td>-</td>
			<td>50 g</td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>0.01</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>Glutathione</td>
			<td>3.2</td>
			<td>-</td>
			<td>20 mg</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>0.6</td>
			<td>10</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>Superoxide dismutase</td>
			<td>0.077</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>Holo-transferrin</td>
			<td>0.062</td>
			<td>-</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>T3 (triiodo-L-thyronine)</td>
			<td>0.0026</td>
			<td>2</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>L-Carnitine</td>
			<td>12</td>
			<td>-</td>
			<td>40 mg</td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>16</td>
			<td>Liquid (1 g/ml)</td>
			<td>20 &#181;L</td>
		</tr>
		<tr>
			<td>D(+)-Galactose</td>
			<td>83</td>
			<td>-</td>
			<td>300 mg</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>183</td>
			<td>-</td>
			<td>322 mg</td>
		</tr>
		<tr>
			<td>Sodium Selenite</td>
			<td>0.083</td>
			<td>1</td>
			<td>280 &#181;L</td>
		</tr>
		<tr>
			<td>Corticosterone</td>
			<td>0.058</td>
			<td>2</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>Linoleic Acid</td>
			<td>3.5</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>Linolenic Acid</td>
			<td>3.5</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>Lipoic Acid</td>
			<td>0.2</td>
			<td>4.7</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>0.02</td>
			<td>3.2</td>
			<td>0.04 mL</td>
		</tr>
		<tr>
			<td>Retinol Acetate</td>
			<td>0.2</td>
			<td>20</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>Retinol, all trans</td>
			<td>0.3</td>
			<td>10</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>D,L-alpha-Tocopherol</td>
			<td>2.3</td>
			<td>100</td>
			<td>0. 2 mL</td>
		</tr>
		<tr>
			<td>D,L-alpha-Tocopherol acetate</td>
			<td>2.1</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
	</tbody>
</table>
Table 1: NS21 Preparation.
Supplementary Video 1: Example video of bioluminescence from well plates. Example video snapshot of bioluminescence from immortalized PER2::LUC in 6 x 96 well plates in the bioluminescence incubator. Wells to be quantified are highlighted in yellow. <a href="http://cloudflare.jove.com/files/ftp_upload/56623/Video_1A_Revision-1.avi">Please click here to download this file.</a>
Supplementary Video 2: Example video snapshot of a bioluminescence recording from PER2::LUC cells under perfusion. A sample area of quantification is highlighted in yellow. <a href="http://cloudflare.jove.com/files/ftp_upload/56623/Video_2B_Revision-1.avi">Please click here to download this file.</a>

Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

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

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

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 ...

flexible-measurement-bioluminescent-reporters-using-an-automated

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

7.8K Views.  MRC Laboratory of Molecular Biology. Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

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

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Immunology and Infection

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Medicine

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Biochemistry

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Developmental Biology

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

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

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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids