The overall goal of this procedure is to obtain enriched mouse embryonic motor neuron culture. The procedure begins with first sacrificing a pregnant mouse, removing the embryos and dissecting the lumbar spinal cord. The next step is to dissociate the spinal cord into a single cell suspension by enzymatic treatment and duration.
Following this, perform the lectin based purification for separating the motor neurons from the cellular remainders. Next, after estimating the total cell number, the cells are plated on pre-coded dishes or cover slips. The final step is to adjust the density of the culture depending on the experimental procedure.
Ultimately, as the cultured cells grow, typical motor neuron morphology can be observed For the success. In preparing culturing mouse and motor neurons, it is essential to test different materials, substances, and solutions. As the cells may react sensitively to any of these in changes, The motor neurons can be cultured in high density for the isolation of RNA or proteins.
However, there's a possibility that the cells will build up active neuronal nets, which lead to the survival independent of traffic support. Therefore, in some cases it might be better to culture the cells in low density for RNA or protein analysis, demonstrating this procedure will be Rebecca Conrad and Terza Japan. Both are graduate students in our laboratory.
Begin this procedure by preparing the working solution of 0.5 milligrams per milliliter poly DL ornithine hydrobromide in 150 millimolar bide buffer with pH 8.35 and sterilize the solution by filtration. Next, sterilize the glass cover slips by flaming them in 100%ethanol. Then let them air dry, cover the surface of the cover slips with sufficient porn H solution overnight at four degree Celsius on the next day.
Wash them three times with sterilized water. Then let them air dry after that. Prepare the working solution of 2.5 micrograms per milliliter.
Laminin in HBSS cover the surface of the porn H coated cover slips with the laminin solution. Then incubate them for at least two hours at room temperature until use. The next step is to prepare a solution of 10 millimolar triss in sterilized water with pH 9.5.
After filtering the solution, add lectin to the mixture to a final concentration of 10 micrograms per milliliter. Then coat the surface of a 10 centimeter cell culture dish with eight milliliters of the lectin solution and incubate it for at least 30 minutes at room temperature. Next, wash the plate with HBS S3 times and store the plate in HBSS until use.
After that, prepare the depolarization solution with 30 millimolar potassium chloride and 0.8%sodium chloride. Then sterilize the mixture by filtration and store it at room temperature. To begin this procedure, thaw horse serum overnight at four degrees Celsius.
Then inactivate it at 55 degrees Celsius for 30 minutes, aliquot into five milliliter portions and store the aliquots at minus 20 degrees Celsius. Thaw one aliquot directly at room temperature before use next store B 27. Supplement in one milliliter aliquots at minus 20 degrees Celsius.
Thaw at room temperature immediately before use. Avoid the freeze thaw cycles aliquot glutamate into 0.5 milliliter aliquots, and store them at minus 20 degrees Celsius. Then prepare a stalk solution of 10 micrograms per milliliter CNTF in sterilized water with 0.1%BSA and store it at minus 20 degrees Celsius.
Finally, mix 46 milliliters of neuro basal medium with 2.5 milliliters of horse serum, one milliliter of B 27 supplement and 0.5 milliliters of glutamate.Directly. Add CNTF to a final concentration of the medium of 10 nanograms per milliliter. Prewarm it to 37 degrees Celsius.
At this point, sacrifice an embryonic 12.5 pregnant mouse and remove the embryos carefully. Add room temperature HBSS to completely cover the embryos and move an embryo under a microscope. Then remove the head and tail and place the embryo backside up with straddled limbs.
Next, fix the embryo with one pair of forceps and remove the outer skin with the other pair of forceps. After that, remove the spinal cord by piercing the forceps under it and lift it up with saw like movements on both sides of the spinal cord. Transfer the isolated spinal cord to a new dish with HBSS.
Open the central channel of the spinal cord on the dorsal side. Next, remove the dorsal root ganglia by removing the enclosed meninges of the spinal cord. Then transfer the spinal cords to an EOR reaction tube filled with one milliliter of HBSS.
Keep them on ice until the preparation is done for spinal cord digestion. Transfer the tube with the spinal cords to a cell culture hood. Carefully remove 700 microliters of the HBSS next at 7.5 microliters of 1%thawed tripsin solution, which was prepared earlier as described in the accompanying manuscript.
And mix them by carefully inverting the tube. Perform trypsin ization for eight minutes at 37 degree Celsius. Then stop the reaction by adding 30 microliters of trypsin inhibitor pipette up and down carefully, 10 to 15 times with a 1000 microliter pipette tip until no more cell aggregates are visible.
After that, repeat the ation with a 20 to 200 microliter pipette. Pipette the cell solution into the HBSS filled lectin plate and disperse the cells by gently rotating the plate. Cover the lectin plate to avoid contamination and keep it on a vibration free surface at room temperature for 60 minutes.
Very gently remove the HBSS and wash the plate four times carefully with prewarm HBSS to remove cell fragments and unattached cells immediately after the last washing step at 500 microliters of the 30 millimolar potassium chloride 0.8%sodium chloride depolarization solution to the plate and incubate it for one minute shake and tap the plate to facilitate the detachment of the lectin bound cells. Fill up the plate with two milliliters of prewarm culture medium. Then transfer it to a 15 milliliter falcon tube.
Count the cell number with a no bower counting chamber Next plate, the cells on the laminin coated cover. Slips or plates. Change the medium on day one and subsequently once every two days by carefully replacing 50%of the old medium with prewarm freshly prepared culture.
Medium shown here is a cross section of an embryonic 12.5 lumbar spinal cord labeled with herst for the localization of the cell nuclei. The arrow indicates the motor neuron columns within the embryonic lumbar spinal cord labeled with eyelet one two antibody. An example of eyelet one two labeled dissociated.
Lumbar spinal cord cells from embryonic 12.5 embryos before and after lectin based pre plating are shown at the left and right panel respectively. The quantitative results of eyelet one two positive cells before and after lectin based pre plating are shown here. Here is an example to show the enriched motor neurons on day zero and day two cells were fixed with 4%paraldehyde and subsequently stained for P 75 NTR.
According to standard procedures, they were then counter stained using hooks to visualize all nuclei after five days in vitro in porn H and laminate as culture substrates and in the presence of CNTF for beta three tubulin staining, the cells have grown out long neurites and displayed the typical motor neuron morphology. With one longer process and one or more shorter processes. Motor neurons play a central role in the body of all vertebrates and invertebrates.
This isolation is extremely useful for the analysis of survival mechanisms of motor neurons. With the isolated cells, you can selectively analyze the activities of other interacting cells, proteins, carbohydrates, and apply pharmacological substances on the survival and neuro growth of these cells.
