Generating a Primary Culture of Astrocytes from a Rat Pup Brain
Transcription
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.
