Nucleofectionとマウス胎児海馬および皮質ニューロンの初代培養

Hippocampal and cortical neurons have been used extensively to study central nervous system neuronal polarization, axon dendrite outgrowth, and synapse formation and function, and advantage of culturing these neurons is that they readily polarize forming distinctive axons and dendrites on a two dimensional substrate at very low densities. This video demonstrates a technique to dissect culture and transfect, embryonic, mouse hippocampal, and cortical neurons via nucleo affection, which enables the expression of fluorescently tagged fusion proteins early in development approximately four to eight hours after plating expression of the fluorescently tagged proteins is maintained throughout the lifetime of the neuron for more than two months in culture. This enables the study of protein function during early developmental events such as axon outgrowth and later events such as synaptogenesis and synaptic plasticity.

First glial cells are isolated from cortical hemispheres of postnatal. Day one to three mouse pops and plated into flasks. After reaching 70 to 100%co fluency, the glial cells are then replated on glass cover slips.

Then hippocampal or cortical neurons are isolated from E 15.5 mice and transfected via nucleo affection with plasmids encoding fluorescent fusion proteins such as red fluorescent protein. The transfected neurons are then plated in imaging chambers for short-term studies. For long-term cultures, glial cells on glass cover slips are inverted over hippocampal or cortical cultures To provide trophic support, individual neurons can then be imaged to show the dynamic localization of fusion proteins throughout axons or dendrites of neurons.

Hi, I'm Eric Dent, an assistant professor of anatomy at the University of Wisconsin Madison. Today, three of my undergraduate students, Chris Wezelman, Jason Bwe, and Derek Lombard are going to demonstrate a procedure for isolating plating and maintaining cultures of cortical and hippocampal neurons for short and long-term culture. These cultures can be used to study the dynamics of fluorescent fusion proteins throughout neuronal development from initial neurite outgrowth through axon and dendrite development and synaptogenesis.

To prepare the chambers for imaging, embryonic, mouse hippocampal and cortical neurons. Begin with 35 millimeter Petri dishes with 15 millimeter holes drilled in the bottom and all burs removed. Melts a three to one paraffin petroleum jelly mixture in a conical tube by placing it in a boiling water bath.

Then use a small paintbrush to coat the underside of the dish around the hole. Be sure to stir the paraffin petroleum jelly mixture between chambers as it will separate. Place the cover slip over the hole.

Once all of the dishes have been prepared, place them in an 80 degree Celsius oven until the paraffin mixture is melted. This should take about 10 minutes. Then remove dishes onto a flat surface and let the paraffin mixture set for at least one minute.

Turn the dishes over and place them under a hood. Turn on the UV light for 30 minutes to sterilize both the insides of the covers and the bottoms of the chambers. Next coat.

The chamber cover slips with poly D lycine for one hour. Then thoroughly rinse the coated cover slips three to five times with sterile filter deionized water to remove all traces of bore buffer After the final rinse, use a vacuum to dry the chambers. These can be used immediately or stored in a tissue culture room for later.Use.

Coated chambers should be used within one week of preparation if long-term cultures will be prepared. Cortical glial feeder layers will need to be started two to three weeks before continuing with the cortical or hippocampal dissections. Place the brains of four P one to P three mouse pops in a dish containing cold dissection medium and place it under the dissecting scope.

Remove the olfactory bulbs, the two cerebral hemispheres and the meninges. Transfer the hemispheres to a new dish containing no media. Using a clean sterile razor blade, mince the cortices as fine as possible.

Then with a plastic pipette, transfer the chopped tissue to a 50 milliliter conical tube containing 12 milliliters of cold dissection.Medium. Add trypsin and DNAs to final concentrations of 0.25%and 0.1%respectively. Incubate in a 37 degree Celsius water bath for 10 minutes with intermittent swirling.

Following the incubation, remove the tube from the water bath and thoroughly clean it with 70%ethanol before bringing it into the tissue. Culture hood. Pipette the cortical tissue up and down.

With a 10 milliliter pipette approximately 10 to 15 times or until most of the chunks disappear, return the tube to the water bath for another 10 minutes with intermittent swirling. Again, thoroughly clean the tube with 70%ethanol and bring it back to the tissue. Culture hood.

Pipette the cortical tissue up and down. As before this time with a five milliliter pipette, add 15 milliliters of warm glial, medium and centrifuge at 1000 RPM for 10 minutes. Discard the supernatant and resuspend the pelleted cells in 20 milliliters of fresh glial medium.

Use a hemo cytometer to count the cells. Then plate five to 7.5 million cells in 15 milliliters of glial medium per 75 centimeter squared flask. Place the cells in the 37 degrees Celsius incubator after one day and every two to three subsequent days in culture, dislodge any loose cells by knocking the flask against the surface of the hood.

Remove the medium along with any dislodged cells and replace it with 15 milliliters of fresh glial. Medium glia can be harvested after one to two weeks of growth in the flasks when they're about to 100%confluent. To prepare individual cover slips coated with glia, begin by placing six nitric acid cleaned and sterilized.

25 millimeter round cover slips in a 10 centimeter dish and using a small paintbrush, place three dots of the three to one mixture of paraffin petroleum jelly. Onto each cover slip in a triangular pattern. Place two dots on the edges of the cover slip to anchor to the dish.

Treat open dishes with UV light for 30 minutes. Coat the cover slips with poly D lysine for one hour. Then wash extensively three to five times with sterile filter deionized water and let dry remove the glial cells from the incubator.

Discard media and wash with five milliliters of prewarm trypsin. Add three milliliters of trypsin to the flask and incubate for one minute at 37 degrees Celsius. Once the cells are detached, add five milliliters of glial medium to stop the trippin.

Remove the GL from the flask by pipetting up and down 10 to 15 times. Then transfer the media to a 15 milliliter conical tube centrifuge at 1000 RPM for eight minutes. Following the centrifugation reus, suspend the cells in 10 milliliters of glial medium.

Then after counting the cells plate five times 10 to the fifth cells in 12.5 milliliters of glial medium per 10 centimeter dish containing the cover slips. Exchange the medium with fresh prewarm glial medium every two to three days the day before the neuron dissection. Remove the glial medium and replace with serum free medium.

Use this glial condition serum free medium later. When flooding, cortical or hippocampal cultures, a C is added to a final concentration of five micromolar. In order to prevent glial proliferation of the nucleo affection reagents per transfection as indicated in the manufacturer's instructions, then warm the solution to room temperature.

Place a dish containing E 15.5 mouse brains in cold dissecting medium. Under dissecting microscope, use a bent tungsten needle to remove both neo cortices. Then using micro forceps, remove the meninges and place the cortices into a new dish of cold dissection medium.

The hippocampus and cortex are visible along the medial side of the cerebral hemisphere with small iris scissors. Carefully remove the hippocampus. Next, remove the remaining cortex.

Repeat the dissection on brains from the entire litter. Place the hippo Camp Cortices in separate 1.5 milliliter micro tubes filled with one milliliter of cold dissection.Medium. Place the tubes on ice After dissecting all of the cortices in hippo Campi.

Add 110 microliters of 2.5%trypsin to the micro fuge tubes containing the tissue, and place the tubes in a 37 degrees Celsius incubator for 20 minutes. Using a P 1000 pipette, remove as much fluid from the tube as possible. Then add one milliliter of plating medium to each tube and wash the tissue by gently inverting the micro fuge tube.

Repeat the wash twice After the final wash, add one milliliter of plating medium. Use a P 1000 pipette to tri Rate the chunks 15 times. Then transfer the supernatant and cells to a new 15 milliliter conical tube containing four milliliters of plating medium, leaving any chunks that remain in a micro fuge tube.

Spin the 15 milliliter tube at 350 RPM for seven minutes with the break off following the centrifugation, discard the supernatant and add 100 microliters of pre-mixed room temperature. Nucle affection solution for each transfection pipette up and down five times to mix next transfer. 100 microliters of the nuclear affection solution and cell mixture to each new micro fuge tube containing an appropriate amount of DNA one to two micrograms of DNA per transfection for long-term cultures will label less than 10%of the neurons in the culture.

While five to 10 micrograms of DNA per transfection will result in a higher transfection efficiency for culture, add the cell suspension and DNA mix to an electroporation vete. Then place the vete into the electro. For mouse CNS neurons, set the nucleo effector program to oh 0 0 5 working quickly, add 500 microliters of the pre-war and equilibrated plating medium to the qve and transfer the cell solution to a new 1.5 milliliter micro fuge tube.

Add enough plating medium to bring the volume of each transfection to one milliliter. After counting the cells plate three to five times 10 to the third cells centimeter squared for young cultures, or five to 10 times 10 to the third cells, percent of meter squared for long-term cultures, generally five times 10 to the fifth or one times 10 to the sixth. Cells are used per transfection to prepare short-term cultures.

One hour after plating flood the 35 millimeter culture dishes with two milliliters of warm carbon dioxide equilibrated, serum free media flooding. The imaging chambers results in a very low serum content less than 0.5%Short-term cultures do not need to be cultured with a glial feeder layer and do not need to be reed. To prepare long-term cultures, add one milliliter of glial condition to serum free medium.

Remove a GL covered cover slip containing three dots of paraffin petroleum jelly and invert it over the 15 millimeter hole in the 35 dish. Then add another one milliliter of medium to feed the long-term cultures. Replace one third of the serum free medium every two to three days.

After five to seven days in culture, cytosine Arab IDE or aee is added to a final concentration of five micromolar in order to prevent glial proliferation. The following paired images of representative living hippocampal neurons show both a differential interference contrast image and a corresponding fluorescent micrograph. Each of these cells has been transfected with EGFP, tubulin and DS red two in PA vectors.

During stage one neurons will initially attach and over a period of a day or so, protrude sheetlike la melo podia, and finger-like Filip podia around the periphery of the cell body. During stage two, the initial protrusions will differentiate into several neurites emanating from the cell body, usually remaining about 30 to 50 microns in length. During stage three stochastically, one of the neurites starts to extend rapidly to become the axon.

The other neurites remain short and are termed minor processes. Most hippocampal and cortical neurons progressed to stage three by two to three days in culture during stage four, which occurs over the next week or two minor processes, thicken, lengthen, and branch to become the dendrites here, these are shown in green. Meanwhile, the axon thins as it continues to grow and branch by two to three weeks.

In culture, dendrites differentiate by forming dendritic spines, small protuberances along the dendrites. Stage five neurons will vary in their shape and size, but most should undergo this series of developmental stages. Transfected neurons may not progress through these stages if overexpression of proteins disrupts neuronal development.

Also, neurons may not progress through all stages of development. If the cover slips are not cleaned properly or the polylysine does not adhere properly, glia can also proliferate extensively, especially if a C is not used. This is a phase contrast image of a two week old culture in which the glia have overgrown the culture in the long-term cultures.

Changing the media too frequently or not frequently enough can also compromise the viability of the neurons. This is a phase contrast image of a culture in which most of the neurons have died and the remaining neurons have shrunken cell bodies and beaded processes For comparison, this is a phase contrast image of healthy hippocampal neurons at two weeks in culture. Note the larger cell bodies and lack of beating in the processes.

Also, at this stage, the dendritic processes have thickened and there is an extensive filigree of axonal processes. Once mastered, embryonic, cortical and dissection, transfection and plating of entire litter of fetuses can be done in two hours if it's performed properly. While attempting this procedure, it is important to remember that hippocampal and cortical neurons are very sensitive to mechanical disruption.

Also, it is important to keep solutions as cold as possible during the isolation of neurons to keep metabolic activity low. Following this procedure, other methods like live cell imaging and physiological recording of activity can be performed in order to answer additional questions such as how fluorescent fusion proteins of interest segregate to different regions of the neuron. If protein localization is associated with changes in synaptic activity, or how the functions of these proteins is affected by different pharmacological treatments.