Obtaining Various Neural Cell Populations from a Rat Hippocampus

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.

Begin the neural dissociation by using sterile razor blades to dice each sample harvested from adult brain tissues into small 30 to 400-milligram pieces on the lid of a six-well culture plate. Then immerse the tissue in 1 milliliter of calcium- and magnesium-free HBSS, and use a pipette to transfer the samples into sterile 2-milliliter centrifuge tubes.

When all of the tissues have been collected, centrifuge the samples, and aspirate the supernatants, and incubate the samples for 15 minutes in a 37 degrees Celsius water bath, inverting the tube several times every five minutes to resuspend the settled pieces of tissue.

At the end of the incubation, add 30 microliters of freshly prepared enzyme mixed tube to each sample, and gently invert the tubes. Then, using a Pasteur pipette labeled 1, dissociate each sample with 30 up and down trituration, taking care to avoid generating bubbles. Incubate the samples for another 15 minutes at 37 degrees Celsius with inversion every five minutes, followed by dissociation of the samples with Pasteur pipette number 2, as just demonstrated.

Then, switch to pipette number 3. Repeat the dissociation and incubate the samples for 10 more minutes in the water bath, with inversion every five minutes. At the end of the incubation, filter the single-cell suspensions through 80-micron cell strainers into 15-milliliter Falcon tubes, washing each strainer with 10 milliliters of calcium- and magnesium-free HBSS to stop the enzymatic reactions. When the last sample has been filtered, spin down the tubes, and aspirate the supernatants.

To deplete the myelin from the cell suspensions, thoroughly resuspend the pellets in 400 microliters of myelin removal buffer, and add the appropriate amount of myelin removal beads to each tube. Pipette the solutions up and down until the cells and beads are thoroughly mixed. Then, incubate the sample at 4 degrees Celsius for 15 minutes. After the incubation, wash each sample in 5 milliliters of myelin removal buffer.

While the cells are spinning, place one column for each sample in the magnetic field of a magnetic sorter, and place a clean 80-micron filter on top of each column. Rinse each filter and column with 1 milliliter of myelin removal buffer three times, collecting all of the flow-through in a waste container.

After the final rinse, place 5-milliliter polystyrene round-bottom tubes directly beneath each column to collect the eluate. Then, aspirate the supernatant from the samples, and resuspend the pellets in 500 microliters of myelin removal buffer.

Immediately apply the cell suspensions to the columns, collecting the cells in the tubes below. Then, rinse each filter and column 4 times with 1 milliliter of myelin removal buffer per wash. After the last wash, spin down the cells and aspirate the supernatants.