The overall goal of this procedure is to study the glial heterogeneity influence on DRG Axon Growth and Cell Deion. This is accomplished by first Turing the glial cells from neonatal rat cortex. The second step of the procedure is to culture and purify the rat embryo DRG neurons.
The third step of the procedure is to add the purified DRG neurons onto the glio cells. The final step of the procedure is to observe the exon growth in cell aion and carry out cell staining and quantification analysis. Ultimately, results can be obtained.
That show DRG neuron growth is influenced by heterogeneous glial cells through time-lapse recording and immunofluorescence microscopy. Hi, I'm from belonging the lab of neuro Imagine at the Department of Neurology in UCRA. Today we'll show you a procedure to study the G cell hydrogenous influence on growth in a new car culture method.
In our laboratory, we use this new method to study the interaction between neurons and the ggl cells. So let's test study. In order to establish a co-culture of glial and dorsal root ganglion neurons or DRGs, we will begin by culturing glial cells under a tissue culture hood Line the bottoms of pre sterilized 24 well culture plates with sterilized round glass cover slips, coat the cover slips in the wells of a six well culture plate with polylysine and incubate for two hours at room temperature.
Wash the cover slips and culture plates twice with distilled water and air dry. Next pipette 200 microliters or two milliliters of dcos modified eagles medium or DMEM with 10%fetal bovine serum or FBS into each 24 well or six well plate respectively. Incubate the plates at 37 degrees Celsius in 5%carbon dioxide the following day.
Sterilize the positive flow dissection hood. Turn on the UV light for 20 minutes and then spray all surfaces under the hood with 70%ethanol and wait 15 minutes before use. Begin with the heads of two P two to P six rat pups that were anesthetized by hypothermia.
Then decapitated at the base of the foram and magnum with operating scissors. Open the cranium along the sagittal suture using iris scissors and peel off the skull. Remove the forebrain and place into chilled levo wit's L 15 and repeat with the second head.
Then working under a stereo microscope. Carefully remove the dura and peer membranes and blood vessels covering the brain. Isolate the cortex and wash several times with L 15 medium.
Use microsurgery scissors to cut the cortex into small pieces. Then incubate an L 15 medium containing 0.125%trips in EDTA at 37 degrees Celsius for 15 minutes. Next, transfer the tissue blocks into a 50 milliliter tube containing 20 milliliters of DMEM with 20%FBS.
Add DS to a final concentration of 10 micrograms per milliliter. Then use a fire polished pasta pipette to agitate the tissue solution approximately 20 times to separate the cells. Finally wash the cells once with DMEM containing 10%FBS determine the cell number with a hemo cytometer.
Then seed the cells at 5, 000 to 10, 000 cells per centimeter square and incubate at 37 degrees Celsius and 5%carbon dioxide. Replace the medium two times per week to isolate purify and culture DRGs. First prepare 24 well plates with polylysine coated glass cover slips as demonstrated earlier.
Add 100 microliters of NVF medium to each cover slip and incubate the plates of 37 degrees Celsius on the following day. Sterilize the positive flow hood as before. Then begin with E 12 to E 15 rat embryos that were isolated from the uterus of a pregnant rat euthanized with carbon dioxide under the stereo microscope.
Isolate the spinal cords with the connected dorsal root ganglia and transfer to 35 millimeter dishes containing chilled levo v's. L 15 medium. Using microsurgery forceps, carefully dissect the connected DRGs from the spinal cords chilled L 15 medium without injuring the DRGs cut the connected axons.
Usually 20 to 30 DRGs are isolated from one spinal cord when all of the DRGs have been isolated from the spinal cords. All the isolated DRGs in 0.125%trips in EDTA and incubate for 20 minutes at 37 degrees Celsius and 5%carbon dioxide wash the cells once DMEM with 20%FBS then add DNAs and pipette several times using a fire polished pasta pipette to form a uniform cell suspension as demonstrated with the glial cells. Perform a second wash in MBF medium count the cells, then seed the cells onto the prepared cover slips in 24, well plates at 5, 000 cells per well incubate overnight to purify the DRG neurons.
18 to 24 hours after seeding. Replace the medium with neuro basal medium containing 20 micromolar five fluoro two doxy uridine 72 hours later. Replace half the medium with neuro basal medium lacking FUDR.
The medium is then replaced every other day. Approximately 20 days after seeding glial cells. They will reach confluence and be ready to co-culture with neurons 24 hours prior to combining the two cell types.
Replace the glial cell medium with MBF medium. Prepare a single cell suspension of purified DRGs as demonstrated previously. Determine the cell density.
Then seed the neurons at 500 cells per centimeter square onto the glial cells. Neuron aian and neurite outgrowth on glial cells can be analyzed at various time points using image analysis software and immunochemistry with cell type specific antibodies. Here are examples of glial cells and DRG neurons cultured separately and as a co-culture glial cells become confluent after 20 days.
In culture. Under a phase contrast microscope, it was evident that different glial cells subpopulations form distinct growth patterns and morphologies. And by immuno cyto chemistry that GFAP positive astrocytes accounted for greater than 90%of the culture.
Using our method of treatment with FUDR for 72 hours, the purity of DRG neurons will reach as high as 99%within six days and will exhibit their unique morphologies with a high density of neurite growth in a co-culture of neurons and glial cells. DRG neuron aian and neurite outgrowth occurred readily on glial cells within four hours after seeding neuron aian and neurite growth were influenced by glial cell subpopulations, which form special growth pattern substructures that could easily be identified under a phase contrast microscope. And by immuno cyto chemistry I'll just to show you how to car culture DRG neurons with clear cells to study the influence of ggl cell heterogeneity on neuro growth.
When doing this session, it is very important to remember to handle the tissue very candidly and to keep the cells in the medium. Never expose them into the air, let em dry. So that's it.
Thanks for watching and good luck with your experiments.
