The aim of this procedure is to visualize fluorescently tagged PKC translocation in living cells. This is accomplished by first preparing SF nine cell cultures or cultures of sensory neurons from a Plegia California. The second step is to express fluorescently tagged pkcs in the cells.
The final step is to image PKC translocation in response to different activators using laser scanning microscopy. Ultimately, results can be obtained that show differential regulation of the different PKC isoforms by second messengers such as diol glycerol through imaging of PKC translocation in real time using live cells. Hi, my name is Carol Ferra.
I'm a research associate in Dr.Wayne Sawn lab at the Montreal Neurological Institute. Today I will be demonstrating live imaging of PKC translocation in Aplysia sensory neurons. This method can help answer key questions in the synaptic plasticity field, such as how are the different PKC isoforms activated during memory formation.
To prepare the SF nine cell monolayer cultures in a tissue culture hood at eight milliliters of grace's insect medium with 30%fetal bovine serum to a 75 centimeter squared cell culture flask then thaw the frozen tube of SF nine cells rapidly in a 37 degree Celsius water bath by moving the tube back and forth for about one to two minutes. Next, transfer the SF nine cells to the cell culture flask and rock the flask gently by hand to distribute the cells evenly. Incubate the cells for one to two hours at 27 degrees Celsius until they have attached.
Then remove the old medium from the flask and replace it with another 10 milliliters of fresh graces insect medium with 30%FBS incubate the cells at 27 degrees Celsius until they are cofluent. To maintain monolayer cultures, remove the old medium from the confluent flask of the SF nine cells and add five milliliters of grace's insect medium with 10%FBS lightly tap on the side of the flask several times to detach the SF nine cells. Then using a 10 milliliter pipette gently pipette the media up and down.
Once spritz in the flask wall while pipetting down, transfer the cells to a sterile 15 milliliter polystyrene tube. Add two milliliters of SF nine cell suspension to eight milliliters of grace's insect medium with 10%FBS in the new 75 centimeter squared cell culture flask with the dilution ratio of one to five. To maintain log phase growth, rock the flask gently by hand.
Next, incubate the cells again at 27 degrees Celsius until they're co fluent. Following incubation, the SF nine cells are ready to use for the expression of fluorescently tagged pkcs as described in the written procedure prior to the expression of fluorescently tagged pkcs Plegia neuronal cell cultures are prepared in matech glass bottom culture dishes as previously described. Two days after isolation of the sensory neurons filter a previously prepared quat of fast green using a syringe and a 20 millimeter syringe filter with a 0.2 micron pore size.
Prepare a solution of plasmid DNA encoding for E-G-F-P-P KC API two in distilled water containing 0.5%fast green at a concentration no higher than 0.4 micrograms per microliter. Filter the plasmid DNA fast green solution using a one milliliter syringe and a four millimeter syringe filter with a 0.2 micron pore size. Then centrifuge the plasmid DNA fast green solution and 16, 110 Gs for 15 minutes using a micro centrifuge.
Next, prepare sharp electrodes for micro injection of neurons using a micro electrode puller. Pull glass micro electrodes using thin wall glass capillaries with filaments inside. Fill each electrode with two microliters of plasmid DNA fast green solution.
Using a micro load pipette tip, microinjection is performed at the microinjection station. The microinjection station consists of a homemade large glass stage to hold the culture dishes on a vibration isolation table. A stereoscope with external halogen illumination, a micro manipulator and a micro electrode holder connected to a pneumatic pump.
The pressure input of the pneumatic pump is connected to a compressed nitrogen tank. Transfer the matech glass bottom culture dish containing the plegia neuron cultures to the micro injection station and insert a micro electrode into the micro electrode holder. View the neurons under a stereo microscope and insert the tip of the micro pipette into the cell nucleus.
Deliver short pressure pulses of nitrogen of 10 to 100 milliseconds in duration and 20 pounds per square inch until the nucleus becomes uniformly green. Incubate the cells for four hours at room temperature and keep at four degrees Celsius until use a Zeiss LSM five 10 inverted confocal microscope with an axia avert. 200 is used for imaging of both SF nine cells and plegia sensory neurons.
Imaging of plegia sensory neurons is demonstrated in this video. Details for imaging of SF nine cells can be found in the written protocol. Use a 30 milliwatts argonne laser with 50%laser output to image PKC tagged with EGFP protein.
Next, attenuate the Argonne laser line to 4%transmission output. Then adjust the pinhole to one, stabilize the culture dish on the imaging stage and avoid movement or vibration. Gently remove one milliliter of culture media from the dish using a 10 milliliter pipette and leave one milliliter total volume.
In the dish. Locate the middle section of the cells where the nucleus can be seen. Set up a time series of 10 to 20 confocal images at time intervals of 30 seconds.
Start the movie and after the second picture is taken, induce translocation by gently adding one milliliter of drug at two times concentration. Add the drug drop by drop on top of the cells continuously for about 30 seconds. Here serotonin is used to induce a fast translocation, which can be visible immediately following drug treatment.
Some drugs induce a slow translocation, which is only optimal five to 10 minutes post-treatment, wash the drug away with two 10 milliliter pipettes, pipetting the wash buffer down gently with one pipette on one side of the dish and pipetting the media up on the other side. If the wash is effective and the effect of the drug is reversible, PKC will revert back to the cytosol within 30 to 60 seconds. Save the movie as an LSM file shown.
Here are the representative results of live imaging of PKC translocation in plegia sensory neurons. E-G-F-P-P KC APL two was expressed in a sensory neuron and a series of 10 confocal images were recorded at time intervals of 30 seconds. In this example, serotonin was added to the dish after 30 seconds of recording.
In this example, live imaging of PKC translocation is shown in SF nine cells. E-G-F-P-P KC APL one and M-R-F-P-P KC APL two were co-expressed In SF nine cells and a series of 10 confocal images were recorded at time intervals of 30 seconds. One two DIOCTYL SN glycerol.
A cell permeable analog of DYL glycerol was added to the dish after 30 seconds of recording. After watching this video, you should have a good understanding of how to express fluorescently tagged pkcs in aplasia sensory neurons, and how to image PKC translocation in live cells.
