The overall goal of this system is to allow the easy observation of bioluminescent reporters under a wide range of extracellular conditions including under constant media perfusion. This method can be used to observe bioluminescence longitudinally from cultured cells and tissues. The main advantage of this technique is that it allows for recording from cells under varying extracellular media and gas conditions which is not possible in most existing systems.
Although here we demonstrate this technique in the context of circadian rhythm experiments, it could readily be applied to other systems that require bioluminescence or fluorescent recording over time scales from days to weeks. To begin the procedure, seed cultured cells on tissue culture dishes or plates. Incubate the cells at 37 degrees Celsius until the monolayers have reached 100%confluence.
Immediately prior to the experiment, use a computer-controlled cycling incubator to alternate between 32 and 37 degrees Celsius every 12 hours for a minimum of 72 hours to synchronize cellular rhythms. Robust synchronization of cellular circadian oscillations both within and between cultures is crucial to experimental reproducibility. We achieve this by applying daily temperature cycles before each recording that synchronize cells by mimicking body temperature rhythms.
Alternatively, pharmacological synchronization can also be quite efficacious. Next, combine 8.3 grams of high-glucose DMEM powder with 900 milliliters of ultrapure water. Stir the solution for 30 minutes to fully dissolve the powder.
Then, add to the solution 50 milliliters of 100 grams per liter glucose solution, 20 milliliters of a pH 7.6, one molar MOPS solution and 10 milliliters of 100 times penicillin streptomycin solution. Stir the mixture for 10 minutes. Then, add 4.7 milliliters of a 7.5%sodium bicarbonate solution and stir the mixture for five more minutes.
Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.6 at room temperature. Add ultrapure water to the solution to achieve a final volume of one liter. Filter the solution through a 0.22 micrometer sterile filter into a sterile flask.
Store the filtered stock medium at four degrees Celsius. Immediately prior to performing the experiment, supplement the desired quantity of stock medium with 10%serum, two millimolar GlutaMAX, 2%NS21 and one millimolar of luciferin. Pass the resulting working medium through a 0.22 micrometer sterile filter.
Use a five molar solution of sodium chloride to adjust the working medium osmolality to 350 milliosmoles. Then, warm the medium to 37 degrees Celsius in a water bath. Next, to begin focusing the bioluminescence incubator camera, unscrew the water-impermeable heated neutral density filter.
In the instrument software, set the exposure time to 0.2 seconds and the kinetic cycle time to 0.3 seconds. Verify that the electron multiplying gain is turned off, then close the setup window and start video recording. Use the focusing cylinder to rotate the camera lens until test items on the appropriate sample shelf are clearly in focus.
Then, stop the video recording. Replace the neutral density filter and remove the test items. Next, use the control panel on the front of the bioluminescence incubator to set the desired oxygen, carbon dioxide and temperature levels for the experiment.
For a humidified experiment, fill the water tray at the base of the incubator. Remove the cells from the tissue culture incubator and replace the culture medium with the pre-warmed working medium. For humidified experiments using small volumes, seal the plates with gas-permeable film or tape.
Place the plates in the bioluminescent incubator. Close the incubator and fasten the door cover to exclude light from the system. Then, in the instrument software, set the acquisition mode to kinetic.
Set the desired exposure time. Set accumulations to one, kinetic series length to the desired number of acquisition cycles and kinetic cycle time to the desired imaging interval. Set a shift speed of 4.33 microseconds, a gain of one and an EM gain of the desired level.
Then, set the auto-save file type to sif or tiff. Name the auto-save file and set its file path. Set the spooling file type to sif or tiff.
Name the file stack and set its file path. Close the setup window and start recording data. Once data collection has finished, import the acquired image stack into image processing software.
Adjust the brightness and contrast for optimal viewing of bioluminescence. Then, select regions of interest and export the mean signal for the selected areas. Copy the data into analysis software for further processing.
To begin the experiment, seed cells on single-channel slides with Luer connectors and a channel depth of 0.6 or 0.8 millimeters. Culture and entrain the cells. Prepare and warm hydrogen carbonate buffered low-glucose DMEM perfusion medium.
Next, cut five two-centimeter sections of silicon tubing with an inner diameter of one millimeter. Cut 1.2-meter, 10-centimeter and 30 centimeter of one-millimeter inner diameter ethylene tetrafluoroethylene tubing. Sterilize the tubing with 70%ethanol and flush the tubing with sterile PBS.
Use the sterilized silicon tubing to connect the 1.2-meter ETFE tubing to a sterile female Luer connector and a sterile elbow Luer connector. Use this assembly to connect the perfusion medium syringe to an empty Luer channel slide. Next, replace the cell culture medium with pre-warmed perfusion medium.
Fill a 20-milliliter sterile syringe with pre-warmed perfusion medium. Fit the 10-centimeter ETFE tubing with Luer connectors using the silicon tubing and connect the tubing to the slide. Flush three to five milliliters of perfusion medium through the tubing and the channel slide.
Then, fit the 30-centimeter ETFE tubing to the cell-containing channel slide. Connect the free end of the 10-centimeter ETFE tubing to the other end of the cell channel slide to form the complete perfusion system. Flush one milliliter of perfusion medium through the tubing and slides to exclude air bubbles.
Transport the perfusion system to the bioluminescence incubator. Secure the perfusion medium-filled syringe in a syringe pump being careful not to disconnect the tubing. Use the syringe pump to flush one milliliter of perfusion medium through the perfusion system.
Enter the syringe diameter on the pump, and set the flowrate to 50 microliters per hour. It is crucial there are no bubbles in the perfusion system at the start of the experiment as these can disrupt the bioluminescence signal. Start the pump and immediately close the bioluminescence incubator door.
Fasten the door cover to exclude light from the system. Acquire and analyze the data as described for static tissue cultures. Six plates of PER2 LUC fibroblasts were entrained using daily temperature cycles and imaged using a bioluminescence incubator.
Images from selected wells were analyzed and the bioluminescence output was quantified. PER2 LUC cells were also imaged under perfusion conditions to evaluate the effects of a Casein kinase inhibitor on PER2 LUC expression. The results were compared to cells treated with the same concentration of the inhibitor under static conditions which revealed substantially greater period lengthening under static conditions.
The magnitude of the period lengthening under perfusion conditions was found to be close to that observed in vivo. After watching this video, you should have a good understanding of how to perform both static and perfused bioluminescence experiments using an ALLIGATOR.
