The overall goal of this method is to stimulate optically controlled substances during flow cytometric measurements. This novel device can help answer key questions in the field of cell signaling by combining the power of optically controlled substances with flow cytometry. For the first time, optogenetic tools can be characterized and analyzed using flow cytometry.
No comparable established method existed until today. To begin, suspend transduced Ramos B lymphocytes at a concentration of one million cells per milliliter and transfer them to a glass FACS tube. Next, exchange the black O-ring of the flow cytometer with a rubber O-ring and apply silicone grease to create a better seal.
Insert the glass tube into a flow cytometer and place the LED prototype around the tube with the lights off. Measure the fluorescence intensity of Dronpa in the GFP channel. The most critical step of the procedure is handling of the glass tubes.
Glass FACS tubes are very fragile and, hence, it is recommended to practice the insertion of the glass tubes into the cytometer prior to doing experiments. Then, switch on the 500 nanometer LED light on the outside of the tube, and continue measuring the flourescence intensity of Dronpa. Increase the intensity of the LED lights until Dronpa fluorescence intensity decreases, in order to estimate the minimal light intensity needed, in order to measure the photoswitching of the Dronpa-Linker-Dronpa protein.
Next, switch on the 400 nanometer LED light of the prototype at the outside of the tube, and continue measuring the fluorescence intensity of Dronpa. Increase light intensity of the 400 nanometer LED lights until Dronpa fluorescence intensity increases. Start by following the design and manufacture instructions in the accompanying text protocol to fabricate the inner and outer layers of the photoswitching LED device.
Insert the 400 and 500 nanometer wavelength LEDs into the channels of the device. Glue them into place and wire them as directed. These specific wavelengths will be used to switch the Dronpa-Linker-Dronpa fusion proteins between their dark and bright states.
Then, assemble the device and make sure it is water-tight in order to prevent leakage prior to powering up the transformer. Following device assembly, harvest Ramos B lymphocytes and suspend them in a density of three to ten million cells per milliliter. Transfer 600 microliters of the cells to a glass FACS tube.
Place them on ice and protect the cells from light until they are measured. Connect the assembled device to a heated water pump and adjust the temperature to 37 degrees Celsius. Then, carefully insert the glass FACS tube into the device and connect it to the flow cytometer.
Record the Dronpa fluorescent intensity in the GFP channel. The design of this optogenetic devices includes a chamber that can be connected to a circulating water supply to maintain the temperature of sample at a constant value. While the device emitted three different wavelengths, only a marginal temperature increase was seen in the FACS sample, when flowing 37 degree Celsius water through the device.
The Dronpa-Linker-Dronpa fusion construct was cloned and transduced into Ramos B cells. When directed into the monomer state by a 500 nanometer light source, the emission intensity decreases. Then, once the light source is switched to the 400 nanometer light, the Dronpa monomer is quickly changed into the dimer state, and the intensity quickly increases.
Using this device, the cytosolic Dronpa-Linker-Dronpa fusion protein was able to be photoswitched multiple times. Switching to the dark state occurs slowly, whereas switching to the bright state happens almost instantaneously. The spontaneous fluorescence recovery of Dronpa in the dark shows that efficient photoswitching to the bright form requires specific illumination and only slowly occurs spontaneously.
The kinetic analysis shows that the photoswitching speed is overall very reproducible and that even after a long incubation in the dark, only about ten percent of the fluorescence is recovered without photoswitching. Following this procedure, any flow cytometric analysis, including calcium flux measurements, can be performed in order to explore the functionality of your chosen optogenetic tool. After it's development, this technique will pave the way for researchers in the field of optogenetics to explore the wiring of cell signaling pathways.
After watching this video, you should have a good understanding of how to establish a photoswitching protocol for your specific optogenetic tool. Don't forget that UV light can be extremely hazardous and precaution, such as protective glasses, should always be worn while performing this procedure.
