The overall goal of this procedure is to introduce cytochrome C protein into the cytoplasm of cells to mimic the release of cytochrome C from mitochondria during apoptosis. This is accomplished by first preparing a mixture of recombinant cytochrome C protein with a dye such as rho domine dextran. This protein mixture is centrifuge to separate small particulates that can clog the needle.
The second step of the procedure is to position the needle above a culture of cells on the microscope stage. Then the cells are micro injected with the cytochrome C containing solution. After microinjection cell survival is monitored, ultimately cells show differential sensitivity to cytochrome C.For example, fibroblasts respond with rapid apoptosis, whereas neurons are remarkably resistant to this insult.
A unique feature of this technique over the treatment of cells with apoptosis inducing drugs is that cytoplasmic micro injection of cytochrome C induces apoptosis without causing mitochondrial damage. This method can help answer key questions in the apoptosis field, such as weather cells differentially regulate apoptosis. Downstream of cytochrome C release Begin by preparing a stock solution of 10 x microinjection buffer.
This solution contains one molar potassium chloride and 0.1 molar potassium phosphate buffer at a pH of 7.4. Long-term storage of this buffer can be at room temperature. To visualize the injection, a fluorescent dye like R Domine dextran needs to be added.
Make a 200 microliter stock of dyed five x micro injection buffer by dissolving Rod Domine dextran powder in 100 microliters of water, and mixing this with 100 microliters of 10 x micro injection. Stock solution. Store this solution in the dark at four degrees Celsius.
It should provide for up to 100 protein solution preparations. Other dyes like fluorescein isothiocyanate. Dextran can substitute prepare cytochrome C stocks by dissolving purified cytochrome C in DNA's RNAs and protease free water to a concentration of 20 milligrams per milliliter.
Divide the stock into 10 microliter aliquots and store them at minus 80 degrees Celsius for long-term storage. Next, prepare a 10 microliter mixture for injection by combining two microliters of five x micro injection buffer with three microliters of water and five microliters of the cytochrome C for a final concentration of 10 milligrams per milliliter. Cytochrome C in one X buffer.
Store the injection mixture on ice and proceed with the injection just prior to microinjection centrifuge the prepared injection buffer to separate any particulate matter that may clog microinjection needles during centrifugation. Turn on the micro injector to allow air pressure to build. Microinjection needles can be made from capillaries or obtained commercially under the microscope.
Use the 10 x objective to focus on the cells to be injected. Cells should only be kept out of the incubator for injection for 30 minutes at most. Now, carefully pipette 0.5 to one microliter of protein mixture from the surface to avoid pipetting any particles centrifuge to the bottom.
Then pipette the protein mix into the blunt end of the microinjection needle. Within a minute, the protein mixture will distribute to the needle tip through capillary action. Then firmly attach the needle to the micro manipulator and position the needle so that its tip passes through the transmitted light of the microscope.
At approximately a 45 degree angle, adjust the position of the needle tip so that it is located directly in the center of the field of view. The needle shadow should be visible in one half of the field of view, indicating that the needle tip is centered above the cells. With the micro injector in position, set it to the continuous flow mode and set the working pressure to 20 to 100 Hector Pascals.
Each needle may require a different working pressure and will likely need adjustment during the micro injection procedure. To maintain a steady flow, now raise the focal plane of the microscope to a position just above the cells. Then lower the needle towards the cells until the needle tip is in focus.
Recenter the needle tip within the field of view and switch to a 40 x objective. Using the fine manipulator knob, slowly lower the needle until it is just above the cells. Next, check the flow of the protein mixture by looking at the red fluorescence of the rod domine.
The protein mixture should be exiting the needle as a thin constant stream. If there is no flow from the needle, try carefully lowering the needle to the bottom of the culture dish to gently rupture the needle.Tip. If the stream is too strong, cells will rupture upon injection.
In this case, the working pressure should be reduced or the needle must be replaced. With flow established. Position the needle towards the cell.
Then with one smooth motion, lower the needle while moving it towards the cell with a second smooth motion. Immediately reverse the direction of the needle, remove it from the cell. A successfully injected cell may swell slightly, and the red fluorescent rod domine can be seen within the cell cytoplasm.
Occasionally a cell will be accidentally injected in the nucleus when moving between cells. Be sure to raise the microinjection needle before moving the stage. Otherwise, the needle can collide with the top of cells.
When this technique is mastered, between 50 to 100 cells can be injected within 30 minutes. Shown here are mouse embryonic fibroblasts injected with cytochrome C.Injection of cytochrome C mimics its release from mitochondria during apoptosis, so after just one hour, these cells have shrunken and show membrane blabbing. Eventually, these cells detached from the culture dish.
In contrast, sympathetic neurons are remarkably resistant to injection with cytochrome C and do not undergo apoptosis after 24 hours. These cells look the same as control on injected neurons. However, XIAP deficient neurons are sensitive to cytochrome C injection five hours after injection, this neuron has died and is no longer visible in the culture.
After watching this video, you should have a good understanding of how to introduce cytochrome C directly into the cytoplasm of cells.
