The overall goal of this procedure is to isolate and culture. Highly purified hippocampal neurons from prenatal mice. This is accomplished by first isolating and dissecting the hippocampus from embryonic mouse tissue.
The second step of the procedure is to dissociate individual cells by mechanical force, followed by trypsin, ization, and careful tation of the brain tissue. Finally, the dissociated cells are counted and cultured using neuro basal media and B 27 supplement. Ultimately purified hippocampal neurons in culture can be used with various molecular techniques such as immunofluorescence.
Visual demonstration of this method is critical in that it's difficult to visualize the iteration process. The tendency is to be too vigorous when separating the individual cells from the brain tissue. Successful general cell ation is required to complete the neuronal culture procedure To generate prenatal pups for neuron harvest.
Schedule breeding between adult mice 19 days prior to the day of neuron isolation. Successful mating can be confirmed by detection of a vaginal plug in the female palpitations or visual confirmation of pregnancy the day prior to neuron isolation. For immunofluorescence applications, coat glass cover slips in a 24 well plate with a light coating of three to one rat tail collagen, one poly de lysine solution spread the solution evenly over the cover slip, rest the coated plates uncovered in a tissue culture hood under a UV light overnight.
The next day, wash the plates with sterile HBSS or store the plates for up to a week at four degrees Celsius in the dark. Soaking an HBSS critical to this operation. Sterilize all the instruments and use only sterile gauze with careful attention to technique.
The dissection and harvest of neural tissue can be completed outside of a laminar flow hood. Begin the tissue harvest by decapitating the pregnant mouse at approximately 19 days Post fertilization. Using dissecting scissors and forceps, create an opening in the mid ventral side of the mouse to completely reveal the mouse's body cavity.
Prenatal pups will be located toward the posterior of the mouse's body cavity and should be easily visible in the uterus with forceps. Open the uterus and remove pups. Decapitate the pups with scissors and place the head on gauze under a dissecting microscope.
To open the cranium, carefully slice through the skin and cranium using a sharp sterile scalpel or dissecting scissors carefully remove the entire brain with forceps and place the brain on gauze. Using a scalpel, remove the cerebellum and incise down the midline of the brain. To separate it into two hemispheres, grasp a small section of meninges surrounding the hippocampus with forceps and pull it gently away to make dissection of the hippocampus easier.
The hippocampus is a curved structure that starts in the distal part of the hemisphere and bends ventrally. The inner concave side is facing a ventricle, is the codal side and is already free. To isolate the hippocampus, gently lift each hippo campi with tissue forceps and transfer them to a 100 millimeter tissue culture dish.
With three milliliters of warmed HBSS under a cell culture hood, brain tissue can be combined from multiple pups using all the sterile precautions possible. Under a cell culture rated laminar flow hood, use a scalpel to gently mince the brain tissue in HBSS. Transfer the mince tissue and HBSS to a 15 milliliter conical tube.
Add 1.5 milliliters of HBSS and 0.5 milliliters of 0.25%tripsin solution to a total volume of five milliliters. Cap and gently invert the tube four or five times. Avoid producing bubbles or the tissue may become trapped on the bubble and not settle to the bottom of the tube.
Incubate the tissue at 37 degrees Celsius for 15 minutes. Inverting tube as before every five minutes after the incubation, carefully pipet away the solution, leaving the tissue at the bottom undisturbed. Now wash the tissue with five milliliters of HBSS.
Three to times allow tissue to completely settle to the bottom of the tube between each wash and carefully remove the wash solution after the final wash. Resuspend the tissue in two milliliters of HBSS Before beginning ation steps. Prepare fire polished pasture pipettes.
Hold a sterile nine inch pasture pipette tip in a flame until diameter of the pipette. Opening is approximately 0.5 millimeters and the edges are slightly rounded. Two large.
An opening will make tation inefficient and too small. An opening will damage the cells, so be careful using a normal sterile nine inch pasture pipette. Gently tri the tissue a total of seven times.
If this process is performed too vigorously, the cells can be damaged and die. Allow larger tissue pieces to settle to the bottom of the tube prior to moving to the next step. Transfer the S supernatant to a fresh sterile 50 milliliter conical tube to the remaining tissue.
Add two milliliters of sterile HBSS and tritrate a total of five times. Using a fire polish pasture pipette. Allow all remaining larger tissue pieces to settle to the bottom of the tube and combine supernatant with the previous supernatant for a total of four milliliters.
Dissociated neuronal cells count the dissociated cells using a hemo cytometer. As a general rule, once cell numbers have been determined, subtract 20%from that final number to account for any cell death that may occur. After plating, mix the cells with neuro basal plating media for 24.
Well plates prepare to add 60, 000 cells per well in 0.5 milliliters of media for 60 millimeter culture plates. Prepare to add 400, 000 cells in three milliliters of media for 100 millimeter culture plates. Prepare to add 6 million cells in six milliliters of media swirl plates gently to distribute cells evenly.
Place neurons in a 37 degree Celsius 5%carbon dioxide incubator overnight. The next day, remove half the volume of media from the cells and replace it with the same volume of neuro basal feeding media. Unlike plating media, this media lacks glutamate and high donor horse.
Serum neurons should be fed every four days by removing half of the old media and replacing it with the same volume of fresh neuro basal feeding.Media. Neuronal processes should begin to be visible on day one and become extensive and branched by day 10. Sometimes contamination by glial cells can occur.
They're easily distinguished from neurons in culture and in this instance, they make analysis of A GFP lc three expressing neuronal process difficult to visualize to reduce glial cell contamination. Treat the culture with cytosine aide before the fourth day of culture at its lowest effective dose. If the culture is contaminated by fibroblastic reactive microglial cells, treat it with FRDR to decrease their proportion.
Hippocampal neurons were grown for seven days and stained with a vital dye to mark active mitochondria and tissue culture cells. The cells were fixed in 4%paraform aldehyde, PBS, and visualized by fluorescent microscopy. The diet itself is non fluorescent until oxidized in the mitochondria.
Active mitochondria can be seen throughout the neuronal processes. Cells from the seven day culture were also fixed with 4%paraform aldehyde, PBS and immuno stain with monoclonal anti tubulin antibody followed by Oregon green labeled goat anti-US secondary antibody and fluorescence visualized by microscopy. Hippocampal neuronal cultures were grown for five days and transfected with G FP lc three beta DNA construct using lipectomy in 2000.
At day seven, the cells were fixed using 4%Paraform Aldehyde, PBS and MICROSOMES with GFP tagged lc three beta incorporated into their outer membrane were visualized using fluorescent microscopy. Acrosome are located throughout the cell body and neurites and are denoted with arrows While attempting to perform this protocol, it's important to remember to perform the procedure quickly but carefully and to be gentle with cells during the iteration process. Paying attention to these variables will greatly enhance your ability to successfully culture.
Mouse hippocampal neurons.
