The overall goal of this procedure is to use a pH sensitive GFP variant to study the spatiotemporal dynamics of Axon guidance receptor trafficking at the cell surface. This is accomplished by first tagging the receptor of interest with the Florin sequence using an appropriate cloning strategy. The second step of the procedure is to characterize the pH sensitivity of the Florin tagged receptor transfected in vitro.
The third step is to electroporated the Florin tagged receptor construct in OVO to achieve expression in the spinal cord. The final step is to section off the electroporated embryos at the desired stage. Ultimately, results can show changes in receptor trafficking at the cell surface in both spatial and temporal domains through immunofluorescence microscopy.
The advantage of this technique over existing methods, such as the use of GFP tech protein constructs, is that the pH sensitivity of pH three winged receptors allows the detection of spatiotemporal changes of the receptor dynamics at the cell surface, both in vitro and in vivo. This method can help answer key question in the Axon guidance field. Indeed, it can help characterizing the spatiotemporal regulation of a cell surface distribution of an Axon guidance receptor, and we know that dysregulation is critical to set the sensitivity of a gross cone to a particular queue.
This method can provide insights into the field of axon guidance within the cheek embryo model, but it can also be applied to other systems such as mouse brain slices. Maintaining culture in combination with life imaging Have prepared cost seven cells in DMEM at 70 to 80%co fluency and the gene of interest cloned into the florin vector. Begin by adding three micrograms of DNA to 200 microliters of 150 millimolar sodium chloride.
Gently vortex the mixture and then spin it down briefly. Then add the appropriate amount of transfection reagent in this case, 10 microliters and vortex. Immediately allow the mix to settle for 10 minutes.
Add room temperature. Next, add 200 microliters of the completed mixture to the cells. Swirl the plate gently and put it back into the 37 degrees Celsius incubator for 48 hours with a media change as described in the text protocol.
Two days later, prepare the microscope chamber. Now transfer the cells to the chamber. Connect a five milliliter syringe to it so that the components can be added directly while the chamber is maintained at 37 degrees Celsius with 5%carbon dioxide.
Before using the microscope, make sure it is equilibrated to avoid mechanical drift during the recording. Next, open the imaging software and select the multidimensional acquisition program and find the transfected cost seven cells with the 40 x objective mark each of their positions in the software where configure the Z stack to a 15 micron acquisition depth. Be aware that the focus can change when adding media to the imaging chamber.
Set the proper phase, the exposure to the GFP filter and proper acquisition timing for an experiment with five fields of interest making image acquisition every 20 seconds over 10 minutes should be sufficient. Start the acquisitions and take five control images in the DMEM pH 7.4 medium. Then pause the acquisitions.
Inject 1.25 milliliters of pH 3.5, complete DMEM to achieve a pH of 5.5 in the culture medium. Mark the event in the software and resume acquiring images for five more time points. During this time, the green fluorescence should gradually disappear.
Then pause the acquisitions. Inject 1.2 milliliters of pH 9.5, complete DMEM to achieve a pH 7.4 in the culture medium. Mark this event in the software and acquire five more time points.
Now, the green fluorescence should reappear at the plasma membrane. Details on handling the eggs prior to electroporation and other preparations are provided in the text protocol. This section begins with creating a window in the egg.
Cover the top of the egg with tape where the window will be made. Then using curved scissors, pierce the shell on its large blunt side. Remove two milliliters of albumin using a needle attached to a five milliliter syringe.
Orient the needle vertically in order to avoid damaging the ylk sack on the top of the egg. Make another hole using the same process. Then from the second hole, cut a window large enough to visualize the embryo and be able to work on it.
At about two milliliters of sterile, warm, modified PBS, this prevents dehydration and increases accessibility. Now inject the DNA and por the embryo. First, dilute the plasmid in PBS to between 0.5 and two micrograms per microliter, but no greater.
Then add fast green dye to a final concentration of 0.025%Load the DNA mixture into a capillary to inject the DNA using an injector. If the capillary's resistance is too large, it could be difficult to inject the embryos. If the resistance is too small, the capillary size could damage the embryo with the loaded capillary puncture, the yolk sack and the neural tube at the coddle side, insert into the neural tube at a shallow angle and fill the lumen from the tail to the head with the DNA mixture.
Next, quickly, place the four millimeter platinum electrodes on either side of the neural tube and electroporated with 50 millisecond, 31 volt pulses every half second. Bubbles should form on the electrodes. Avoid the heart or large extra embryonic vessels.
When the electroporation is finished, use a needle to remove two milliliters of albumin. Then seal the window and the blunt side hermetically with tape. Return the eggs to 38.5 degrees Celsius until they develop to the desired stage.
Then follow the procedures for embedding and cryosectioning given in the text protocol, the use of Florin provides precise spatiotemporal resolution of transmembrane receptor sorting at the plasma membrane. For demonstration plex in a one was tagged with Florin. It is a guidance receptor that mediates the signal of se Forin three B.The construct was expressed in vitro.
In CO seven cells and live cell imaging was performed at a physiological pH. The receptor was mostly at the plasma membrane, thus confirming the detection of only the cell surface pool of the receptor. After innovo electroporation, it was found that plex in a one distributes at the surface of the growth cone upon floor plate crossing.
At the pre crossing stage plex in a one was not detected at the cell surface. This suggests SEMA four and three B Axon signaling is limited to the postcrossing stage. While attempting this procedure, it should be noted that the use of pH flu is primarily suited for the monitoring of life preparation.
The use of pH flu in fixed tissue requires a particular care during and after fixation. Since the confirmation of pH flu may only be temporary, thus the pH of all solutions must be kept above seven, and the observation should be performed shortly after fixation After its development. This method paves the way for researcher in the field of Axon guidance and cell migration to explore the regulation of cell-surface distribution of a guidance receptor in different animal models such as cele against drosophila and mouse.
