Selectively Removing Microglia from a Rat Cerebellar Granule Neuron Culture
내레이션 대본
Begin this procedure by collecting the cerebella from four to seven-day-old rat pups. Place them immediately in a Petri dish containing five milliliters of solution A on ice. Then, remove excess solution from the cerebella and place the tissue in the Petri dish lid. Next, chop the tissue finely with a well-flamed razor blade in at least three different directions.
Subsequently, add the chopped tissue to solution B. Place it in a water bath at 37 degrees Celsius for five minutes, and shake it gently every couple of minutes. Then, add 20 milliliters of solution D to the tube to neutralize the trypsin. Shake and centrifuge at 65 g for five minutes. Afterward, pour off the supernatant. Resuspend it in four milliliters of solution C.
Triturate the sample 10 times with each of the three flamed glass pipettes of decreasing diameter until the suspension is as homogeneous as possible. Next, slowly and gently add a few drops of this homogenate on top of the BSA-EBSS. If it starts to sink through the BSA-EBSS, triturate more and add a little more solution C. The homogenate should sit in a layer on top of the BSA-EBSS.
Spin it at 100 g for five minutes, and do not shake. Then, remove the supernatant and resuspend the pellet in one milliliter of MEM medium. Count the cells using a hemocytometer, and plate them at 800,000 cells per coverslip in 500 microliters of MEM medium. After that, place them in an incubator at 37 degrees Celsius with 6% carbon dioxide. Then, make a solution of MEM medium containing 10 micromolar of the cell cycle inhibitor, AraC.
For each coverslip, retain 250 microliters of the old medium in a fresh 24-well plate and aspirate the rest. Add 250 microliters of normal MEM medium to wash the cells. Subsequently, add 250 microliters of old medium back to the cells, and top up with 250 microliters of AraC-containing medium.
In this procedure, add pure LME to five milliliters of MEM medium to give a final concentration of 150 millimolar LME. Return the solution to pH 7.4 using acid-base solution. Then, sterile filter it using a 28-millimeter PES with 0.2-micrometer syringe filter.
Next, dilute the LME solution to a concentration of 50 millimolar in MEM medium. Then, place it in a water bath at 37 degrees Celsius for 10 minutes, along with normal MEM medium for control cultures. After 10 minutes, remove 250 microliters of the MEM medium from each CGC culture. Then, retain the media at 37 degrees Celsius.
Next, treat the cells with MEM medium containing two times LME or with pre-warmed MEM medium without LME for control cultures. Incubate the cells at 37 degrees Celsius in a humidified atmosphere with 6% carbon dioxide for one hour. Afterward, wash the cells twice in fresh pre-warmed MEM medium to remove the LME-containing media.
Then, replace the retained culture medium with an equal amount of fresh pre-warmed MEM medium. Lastly, incubate the cultures in 6% humidified carbon dioxide at 37 degrees Celsius.
