To begin, take neurons grown in T25 flasks for co-culture. Replace the media with five milliliters of versene solution containing trypsin and Dnase I.Add 0.5 milliliters of fetal bovine serum to neutralize the trypsin and Dnase I.Transfer cells to a 15 milliliter conical tube. Wash the flask with five milliliters of complete neurobasal media once to maximize the collected neurons.
Then, centrifuge the cell suspension at 300 G for five minutes at four degrees celsius. After discarding the supernatant, gently resuspend the cell pellet in two to three milliliters of complete neurobasal media. Seed 100 microliters of the cell suspension to each well to obtain 75, 000 neurons per well, between eight to 10 days of mixed glia cultivation.
Using a 10 milliliter pipette, gently wash the T75 flasks with glia culture media to collect microglia and transfer the cells and media into a sterile 50 milliliter conical tube. Centrifuge the cell collection at 300 G for five minutes at four degrees celsius. Once the supernatant is removed, gently resuspend the pellet in two milliliters of complete neurobasal media.
From the 96-well neuronal culture plate, remove 100 microliters of neurobasal media and add 100 microliters of 250, 000 cells per milliliter cell suspension. In co-culture round and large microglia were observed on top or between neurons and neurite processes.
