To begin, take the brain isolated from the euthanized mouse pups and place it in a three-centimeter Petri dish containing cold MEM with EBSS and 2X PSN. Under a dissecting scope, separate brain hemispheres, remove and discard the midbrain, hippocampus, and meninges. Place both cortical hemispheres in a 50-milliliter conical tube containing five milliliters of MEM EBSS with 2X PSN, and keep on ice.
In a tissue culture hood, aspirate the media from the 50-milliliter conical tube using a pipette, leaving the cortical tissue pieces at the bottom. Add 10 milliliters of pre-warmed dissociation media with a coated glass pipette, and triturate the tissue 10 to 20 times. Transfer the suspension to a 50-milliliter beaker containing a small stir bar and place it on a stir plate.
Then remove the beaker, set it at a 30-degree angle for three minutes to allow the tissue to settle, and transfer the cell suspension to a new 50-milliliter conical tube on ice. Centrifuge the 50-milliliter conical tube at 1000G for 10 minutes at four degrees Celsius. Replace the supernatant with glial growth medium and count the cells using a hemocytometer.
Plate the cells at a density of one million in 10-centimeter cell culture-treated dishes and incubate for 24 hours. Then replace the media with fresh glial media and allow the cells to reach 100%confluence within two weeks before plating them for experimentation. Plate 100, 000 glial cells per well in six-well plates and allow them to reach 80%to 100%confluence for in vitro prion infection.
Expose 20%diluted brain homogenates and PBS to ultraviolet light for 30 minutes. Aspirate the media from the Glia to be infected and add 1.5 to 2 milliliters of glial growth media with 0.1%normal or prion brain homogenate per well. After 72 hours, remove the media, wash the cells with PBS and add fresh glial growth media.
Carefully aspirate the adipose tissue in approximately one milliliter of HBSS in 0.25%trypsin solution, using a serological pipette. Transfer the tissue to a four-centimeter Petri dish containing two milliliters of DMEM F-12 media with Dnase-1, collagenase and Dispase mixture. Then cut the adipose tissue into small pieces using scissors and incubate the mixture at 37 degrees Celsius for 1.5 hours.
Transfer the tissue to a 50-milliliter conical tube, triturate the tissue, and pellet the stromal vascular fraction, which will appear red. After washing the pellet with sterile PBS and centrifuging for three minutes, resuspend the pellet in one milliliter of ADMSC media. Afterward, pipette the cell suspension through a 40-micrometer strainer into a sterile 50-milliliter conical tube, to remove non-dissociated tissue.
Add nine milliliters of ADMSC media to a 10-centimeter cell culture-treated dish. Pipette the strained cell suspension into it and incubate at 37 degrees Celsius with 5%carbon dioxide. Change the media the next day and passage the cells once they attain 80%to 90%confluency.
After the cells have undergone two to three passages, resuspend them in three to 10 milliliters of media and count using a hemocytometer. Plate 100, 000 cells per well in six-well plates containing ADMSC media and incubate overnight, as shown earlier. The next day, prepare the ADMSC media for cytokine-stimulated cells with either 10 nanograms per milliliter of TNF-alpha or 200 nanograms per milliliter of IFN-gamma.
Create ADMSC media with a final concentration of 0.1%prion-infected or normal brain homogenate for control samples. Aspirate the old media, add 1.5 milliliters of cytokine or brain homogenate-containing media into the corresponding wells in triplicate, and return plates to the incubator. Then wash the cells with PBS and isolate RNA by adding 350 microliters of lysis buffer with 1%beta-mercaptoethanol to each well.
Remove cell lysates using a cell lifter and reverse transcribe 25 nanograms of RNA per sample and amplify complementary DNA. Plate BV2 microglia at 50, 000 cells per well or primary mixed glia at 100, 000 cells per well in a six-well plate. Treat BV2 cells with media containing 0.1%prion-infected or normal brain homogenate and incubate for 72 hours.
Following incubation, wash the cells two times with PBS to remove the remaining brain homogenate. Add fresh media to the cells and return the plate to the incubator. To stimulate ADMCs, treat them with 10 nanograms per milliliter of TNF-alpha for 24 hours before co-culturing with BV2 or mixed glia.
Wash the stimulated ADMCs with PBS three times and spin the ADMSC suspension as demonstrated earlier. Replace the media on BV2 cells or mixed glia with two milliliters per well of ADMSC media, and place inserts for six-well plates with a pore size of 0.4 micrometers into half of the wells. Add two milliliters of ADMSC media to each insert and add an additional two milliliters of media to wells that do not receive inserts.
Upon pelleting, resuspend and counting the ADMCs, add 50, 000 to 100, 000 cells to each insert and incubate them. At the end of the incubation, remove and discard the inserts or replace them in a new six-well plate and wash the inserts twice with PBS. Add the buffer provided by the RNA isolation kit to the mixed glia or BV2 cells and scrape the wells.
