Generating a Primary Culture of Astrocytes from a Rat Pup Brain

Transfer the cortex into a 15-milliliter tube containing three milliliters of cold PBS, and homogenize the tissue by pipetting up and down 10 to 15 times with a one-milliliter tip. Centrifuge the cells at 500 g for five minutes. Discard the supernatant, and resuspend the pellet in three milliliters of cold PBS by pipetting with a one-milliliter tip.

Centrifuge once again and resuspend the pellet in two milliliters of complete DMEM, supplemented with 10% FBS and antibiotics. After transferring the homogenate to a two-milliliter tube, pass the homogenate through 21- and 23-gauge needles to make a suspension of single cells. Pour the cell suspension prepared from one cortex into a 60-millimeter Petri dish containing three milliliters of complete DMEM, and lightly shake the Petri dish so that the suspension is uniformly distributed. Incubate the cells in humidified incubator with 5% carbon dioxide and 95% air at 37 degrees Celsius.

To promote astrocyte growth, remove the old media, and wash the cell with pre-warmed PBS to remove the loosely attached glial cells and traces of FBS, once they attain 70% to 80% confluency. Then, trypsinize the underlying layer of astrocytes by adding one milliliter of pre-warmed trypsin solution, and incubate at 37 degrees Celsius for two to five minutes.

Use a microscope to check if the cells begin to detach, and add four milliliters of complete DMEM. Collect the cell suspension, and transfer it to a 15-milliliter tube. Then, centrifuge the tube at 500 g for five minutes, discard the supernatant, and resuspend the pellet in one milliliter of complete medium. Count the cells using a hemocytometer.

Next, prepare the culture dish, and add culture medium. Replate the cells at a density of 10⁴ cells per square centimeter in five milliliters of fresh, complete DMEM, and wash the cells with complete DMEM before each media replacement.