The goal of this procedure is to obtain pure axonal preparations suitable for examination by conventional, biochemical, or immuno cyto chemical techniques. This is accomplished by first culturing embryonic dorsal root ganglion cells on culture inserts bearing a porous membrane filter as the DRG neurons extend axons. Some of the axons grow through pores in the filter and extend on its bottom surface while the cell bodies remain isolated on the top surface After the desired experimental manipulations, the axonal preparation is obtained by physically removing the cells on the top side of the filters while keeping the axons that grew on the bottom side.
Ultimately, the isolated axons can be lyed for biochemical analyses or fixed for immunochemical examination By producing pure axonal samples suitable for analysis by conventional biochemical techniques. This procedure has proven to be very useful to investigate the physiology and pathophysiology of axons. As you will see, the technique is relatively simple to implement and can be adapted to particular experimental settings.
We are going to demonstrate its use in studying axonal degeneration mechanisms The day before plating. Begin by coating the filters under a sterile tissue culture hood. First place, 24 millimeter filter inserts in a six well receiving plate.
Then add two milliliters of one milligram per milliliter, poly de lycine in water to the bottom compartment and one milliliter to the top, and incubate the inserts for one hour at room temperature. Her next aspirate the poly de lycine being sure to remove the remnant of liquid that becomes trapped directly under the filter. Then rinse once with water after aspirating the water, close the lid and leave the plates to dry in the hood overnight.
The next morning, mix and warm a laminate and water solution at 37 degrees Celsius. Incubate the inserts in the laminin solution for one hour in a cell incubator at 37 degrees Celsius using standard volumes. After aspirating.
The laminin add 0.1 milligrams per milliliter, collagen in water and incubate for one hour at room temperature, then aspirate the collagen and wash once with sterile water. The filters are now ready to receive culture media and DRG explan after, after dissecting and decapitating an E 13 mouse embryo as described in the text protocol. Place the embryo on its side in a Petri dish filled with L 15 media while observing through a dissecting microscope, make an incision along the lateral sides to discard the chest, belly limbs and tail.
Place the back containing the spine ventral side up and remove all of the visceral organs using small spring scissors. Expose the entire spinal cord by cutting the spinal column from its ventral side along the midline. Hold the carcass down with one pair of number three forceps and use a second pair to hold the spinal cord by its most rostral aspect.
Now, slowly and gently pull the cord away from the vertebral cavity. As the DRGs are exposed, use ultrathin number five forceps to pinch them off the spinal cord. Place the detached DRGs on the bottom of the dish using a 200 microliter pipette tip that has been cut to enlarge.
Its opening. Aspirate the collected DRGs and place them in a tube on ice. Supplement the bottom compartment media with 15 nanograms per milliliter of NGF while keeping the top compartment NGF free.
Next, add complete DRG media at 37 degrees Celsius to the coated inserts using standard volumes. Then using a 1000 microliter pipette with a tip trim to enlarge its opening. Set the pipette at 800 microliters.
Then transfer approximately 200 microliters of media from the top side of the filter to the DRG containing tube pipette a small volume up and down in order to resus suspend the D RRGs that have sunk to the bottom. Once Resuspended aspirate the whole volume, now transfer the DRGs to the filter by submerging the pipette tip in the middle of the insert and expelling the DRGs. Maintain the cultures in a cell incubator at 37 degrees Celsius.
Changing the media every two to three days to change the media. Aspirate the top compartment first, followed by the bottom compartment. Add fresh media in standard volumes starting with the bottom compartment to study the effect of trophic factor withdrawal on axons.
Wait two days to allow for axonal extension. Next, remove the media from the top and bottom compartments. Then to sequester any remaining or endogenously produced and GF add media that lacks NGF and contains anti NGF antibodies.
Return the plates to the cell incubator at 37 degrees Celsius to allow NGF withdrawal induced degeneration to proceed for the desired amount of time to study while arian degeneration. Use filters containing DRG explan maintained in NGF for two days. Use a cell scraper to scrape DRGs from the top side of the filter.
Ensure the complete removal of the neuronal cell bodies by moving the scraper back and forth in the x and y directions and in circles. Confirm the success of the scraping by checking that no X plants remain attached to the filter. Next, discard the media containing the floating explants from the top compartment and add one milliliter of prewarm complete DRG media containing 10 nanograms per milliliter and gf.
Then return the plate to the cell incubator at 37 degrees Celsius to allow wallerian degeneration to proceed for the desired amount of time to extract protein from the axonal samples. Begin by filling 1.5 milliliter collection tubes with 75 microliters of lamb leaf sample buffer and place the tubes on ice. Wash the filter containing the DRG explan in PBS and scrape the top side of the filter as before.
After removing the insert from the plate, use a cotton tip applicator to clean the top side of the filter. Then aspirate any extra PBS from the top and bottom sides. Next, place the insert upside down on a bench and use a sharp scalpel to cut the filter out of the insert.
Then holding the filter with forceps. Bottom up. Use scissors to make a cut from the circumference to the center of the filter.
Now place the filter on top of the collection tube and with forceps, press the center of the filter down the tube. While doing this, a funnel will form push the funnel shaped filter to the bottom of the tube so that it is submerged in the laly sample buffer. Now complete the protein extraction by covering the tubes and placing them in boiling water for four minutes.
Remove the filter using forceps and place it upside down into the top of the tube and centrifuge it briefly. When done, discard the dried filters for immunofluorescence. Wash the inserts in PBS in six well plates.
Then fix the inserts in freshly prepared 4%paraform aldehyde in PBS for 10 minutes at room temperature on a shaker after fixation, scrape and clean the top side of the inserts to ensure that only axons that grew exclusively on the bottom surface of the filter will be stained. Then perform standard immunofluorescence as described in the text protocol. Once all immunofluorescence incubations are completed.
Pour off the PBS from the last wash. Clean the top side of the filter with a cotton tip applicator and aspirate any remaining liquid from the top and bottom sides. Place the insert upside down on the bench and use a sharp scalpel to cut the filter out of the insert.
Then holding the filter with forceps downside up. Place it on a microscope slide. Use fluorescent compatible mounting media to place a cover slip over the filter and let the filter dry flat in the dark at four degrees Celsius.
Finished by examining and capturing images using a fluorescent microscope. The sequential coating of filters with polylysine, laminin and collagen results in-plants that grow more and longer axons compared to those grown with polylysine alone or polylysine and collagen under the same substrate. Coating conditions.
DRG Explan maintained on filters show considerably more exuberant axonal growth than X plants grown on plastic. The axonal preparation obtained after scraping the top side of the filter is devoid of cell nuclei demonstrating that cell bodies are excluded from the bottom side of the filter shown. Here are examples of axons that originated from approximately 17 explan, which extended through the filter pores to grow on the bottom side of a 24 millimeter filter.
Immuno blots of lysates collected from filter tops versus bottoms demonstrate that histone H three a nuclear marker is confined to the filter tops. The total protein content was normalized across the different samples. The NGF deprivation experiments are demonstrated in these images at different magnifications of axonal preparations from explan maintained in NGF or deprived of NGF for 12 or 36 hours.
The wall Arian axonal degeneration studies are shown in these images of axonal preparations from intact explants or three to 7.5 hours after transection, as in other models of wallerian degeneration. Five millimolar NAD plus protects from transection induced degeneration Once mastered. This technique is robust and easy to reproduce.
It allows for the generation of large quantities of reliable axonal preparations Since first described by twist and colleagues in 2001. Variations of this technique have been used to explore many aspects of axon and dendrite physiology. Refer to the citations in the text protocol to discover other applications of this technique.
