Generating a Primary Culture of Astrocytes from a Rat Pup Brain

Add a cortex from a rat pup brain to a tube with a buffer.

With a pipette, gently move the cortex up and down to break it into small tissue pieces.

Centrifuge and discard the supernatant.

Resuspend the pieces in a culture medium.

Pass the pieces through needles of decreasing diameters to obtain a single-cell suspension.

Transfer the cells to a polypeptide-coated plate containing the culture medium and incubate.

Astrocytes strongly adhere to polypeptide-coated surfaces, facilitating their growth over other cells.

Remove the medium and wash with a buffer to remove any loosely attached cells.

Treat with a digestive enzyme to detach the astrocytes from the plate.

Add the medium to stop the enzymatic activity.

Collect the supernatant in a tube, and centrifuge.

Discard the supernatant and then resuspend the cells in a fresh culture medium.

Place the cells into a plate and incubate to facilitate the multiplication of astrocytes.