The overall goal of this experiment is to observe cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This method can help answer key questions of how cells communicate with each other through the Notch signaling pathway, particularly of how cells transmit the oscillatory information to each other. The main advantage of this technique is that we can control and monitor gene expression dynamics with very high precision.
Demonstrating the procedure will be Akihiro Isomura, a research fellow from my laboratory who developed this new technology. To transfect plasmid vectors of Tol2-based optogenetic modules together with the transposase expression vector into C2C12 cells, first count trypsinized cells with a cell counter. Plate 50, 000 C2C12 cells per well in a 12-well plate one day before transfection.
Then, co-transfect 0.375 micrograms of Tol2-based optogenetic vectors, 0.125 micrograms of a drug selection vector, and 0.5 micrograms of the transposase expression vector by using the lipofection reagent. Trypsinize the transfected cells, and plate them into 100-millimeter culture dishes one day after transfection. Exchange the culture medium for the transfected cells to culture medium supplemented with 100 milligrams per milliliter hygromycin.
Culture the cells for three days to eliminate untransfected cells. Note that medium change is not necessary. Next, trypsinize the transfected cells, and purify a population of cells expressing fluorescent proteins for selection by sorting the receiver and photo-sensitive cells, as detailed in the text protocol.
Set up two types of incubators with or without light sources for a light-induced condition or a dark condition. Use a light meter to set up the light intensity of a blue-LED transilluminator. Measure the light intensity after closing the door.
Program timings and duration of light illumination by loading a control script to a single-board microcontroller that allows programmable schedules for illumination. Count the trypsinized cells with a cell counter, and plate 100, 000 photo-sensitive sender cells carrying pAI218 and pAI170 on 35-millimeter-diameter plastic culture dishes. Set the dishes in separate incubators for dark and light conditions.
After setting up the dishes, keep the doors of the incubators closed until collecting the cell lysates. One and a half days after plating, start light illumination. Don't expose cells to uncontrolled light to avoid undesirable photo-stimulation.
So, do this step without opening the door. Note that opening the door here is only for demonstration. About two days after plating, move sample dishes from incubators to ice in 30-minute intervals, and start preparation of cell lysates for further analysis.
To monitor the cellular responses upon optical stimulation by PMT in real-time, first trypsinize and count the numbers of sender and receiver cells with standard methods. Prepare a one-milliliter total volume suspension of 25, 000 receiver and 125, 000 sender cells. Plate the mixed cells in each well of 24-well black plates with culture medium containing one-millimolar luciferin.
Analyze the cells on a plate reader for the luciferase assay to confirm that the output signals are not too high for the recording system. Next, set the plate on the recording system, and start a recording program. For example, start light illumination at 18 hours after setting the recording.
For real-time imaging of single-cell responses under the control of optogenetic perturbation, first plate 50, 000 receiver and 250, 000 sender cells onto 27-millimeter-diameter-glass-base 35-millimeter-diameter dishes. Ensure that mixing ratios and total cell number are correctly adjusted. One day after plating, exchange medium with two milliliters of the recording medium.
Set the glass-base dish on an inverted microscope equipped with an environmental chamber at 37 degrees Celsius and 5%carbon dioxide. Set up a cooled CCD camera. Ensure that the temperature of the CCD sensor is cooled down to the destination value.
Set parameters for multi-dimensional time-lapse window in automatic acquisition software to capture images with five-minute intervals and more than 288 times. In the multi-dimensional time-lapse window, select a luminescence channel. Ensure that the readout mode is set to the slow 50-kilohertz readout mode, which is critical for reducing readout noises to detect weakly emitting bioluminescent light.
In the multi-dimensional time-lapse window, select fluorescence channels. Ensure that the readout mode is set to the fast one megahertz mode to reduce the time required for fluorescence imaging. Finally, set two-by-two binning with 400-millisecond exposure.
Next, select a journal tab to set up schedules to stimulate the cells with periodic blue-light illumination. Click the plus button to add a new journal setup. Select a journal file from a journal box, choose multiple time points, and set values in initial point and interval boxes to schedule for stimulation.
Ensure that the specified journal file includes sequential protocols for selecting illumination, shutter opening, delay, shutter closing, and delay. Next, set up schedules to stimulate cells with periodic blue-light illumination. Finally, start time-lapse recording by clicking the Acquire button.
Finally, extract single-cell traces of luminescence channels from time-lapse movies, as described in the text protocol. Representative results of optogenetic sender-receiver assays are shown here. The photo-inducible sender cells produced oscillatory patterns of Delta ligand expression in the presence of periodic blue-light illumination, as expected.
When receiver cells are co-cultured with the photo-sensitive sender cells and exposed to repetitive light illumination, a real-time bioluminescence recording system detects cyclic responses of receiver cells in various conditions of mixing ratios. Moreover, time-lapse microscopy with repetitive light illumination also reveals the synchronized responses of receiver cells at the single-cell level. After its development, this technique paved the way for researchers in the field of cell biology to explore how dynamic information of gene expression is transferred in cell-cell interactions.
