Obtaining Various Neural Cell Populations from a Rat Hippocampus
Transcription
Take perfused rat hippocampus fragments.
Incubate with papain to digest the tissue's extracellular matrix and loosen cells, including neurons, astrocytes, and microglia.
Add DNase and mix.
Mechanically dissociate the tissue using pipettes of progressively decreasing diameters to obtain a single-cell suspension. Incubate for the DNase to degrade contaminating DNA.
Pass the suspension through a strainer, removing debris.
Add buffer containing divalent ions to stop the enzymatic activity.
Centrifuge and remove the enzyme-rich supernatant.
Now, add buffer and anti-myelin magnetic beads. Incubate for the beads to bind the myelin debris.
After incubation, centrifuge and remove the supernatant.
Resuspend the mixture in buffer and load it onto a magnetic separation column containing ferromagnetic spheres placed in the magnetic field of the magnetic sorter.
The magnetic field retains magnetic bead-bound myelin debris in the column while myelin-free cells pass through.
Wash the column with buffer and collect the flow-through containing neurons, astrocytes, and microglia.
