A Technique to Isolate Nuclei from Non-neuronal Cells in the Hippocampal Dentate Gyrus

Take dentate gyrus from a mouse brain in a cold homogenization buffer.

Mechanically dissociate to release neurons, glia, and stem cells.

The buffer's mild detergent permeabilizes the cells, releasing nuclei and other organelles.

Filter the homogenate to remove tissue debris. Then, centrifuge, remove the organelles, and resuspend in the homogenization buffer to lyse any intact cells.

Centrifuge again, remove the supernatant containing released organelles, and resuspend the nuclei in a wash buffer.

Filter to remove any debris, centrifuge, and remove the supernatant.

Introduce a fluorescent DNA stain to label all nuclei and a fluorophore-conjugated antibody that binds to the neuronal nuclear antigen, or NeuN, labeling the neuron nuclei.

Using fluorescence-activated nuclei sorting, identify aggregates with higher DNA stain fluorescence intensity and higher scattering properties, separating the single nuclei.

Analyze the single nuclei, separating the NeuN-positive neuronal nuclei and isolating the NeuN-negative nuclei belonging to the glia and stem cells.

Homogenize the tissue with 10 strokes of the loose A pestle, followed by 15 strokes of the tight B pestle. Transfer the homogenate into a pre-chilled 15-milliliter tube. Rinse the Dounce homogenizer with 1 milliliter of cold HB, and combine it with the same tube.

Add 3 milliliters of HB to the 15-milliliter tube, and incubate for 5 minutes on ice. Mix this nuclei suspension twice by inverting the tube gently. Using 0.5 milliliters of HB, pre-wet the 70-micrometer strainer cap placed over a 50-milliliter test tube. Then, strain the nuclei suspension before washing the cell strainer with 0.5 milliliters of HB.

Next, remove the cell strainer, and centrifuge the test tube in a swing bucket centrifuge at 500 g for 5 minutes at 4 degrees Celsius. Discard the supernatant. Gently resuspend the pellet in 4 milliliters of HB using a P1000 pipette.

After five minutes of incubation on ice, centrifuge the suspension at 500 g for 10 minutes at 4 degrees Celsius. Discard the supernatant, and resuspend the pellet in 3 milliliters of wash media. Use 0.5 milliliters of wash media to pre-wet a 35-micrometer strainer cap over a 50-milliliter test tube. Then, strain the nuclei suspension by pipetting 0.5 milliliters of suspension at a time using P1000 pipette.

After washing the strainer cap with 0.5 milliliters of wash media, place the tube on ice. Transfer the filtrate to a new 15-milliliter tube, and centrifuge it for 5 minutes and 4 degrees Celsius at 500 g. Discard the supernatant, and resuspend the pellet in 3 milliliters of wash media.

Repeat the centrifugation, and resuspend the pellet in 1 milliliter of wash media with the mouse anti-Neu and AlexaFlour488-conjugated antibody and 1 microgram per milliliter DAPI. Incubate the reaction for 45 minutes on ice in the dark.

For Fluorescence Activated Nuclei Sorting or FANS, transfer the immunostained nuclei suspension to a 5-milliliter test tube, and place it on ice until the start of the flow cytometry procedure.

Vortex the samples for three seconds with mild intensity before placing the tubes into the FACS instrument.

To acquire the data from stain nuclei suspension, set the gates in DAPI height and the DAPI area to exclude the cell debris and the aggregated nuclei. Then, set the gates in the log side-scatter or SSC area and the log forward-scatter or FSC area to separate the single nuclei from the remaining DAPI-stained aggregate or cell debris.

Then, isolate the NeuNAF488-negative population by setting the gates for the anti-NeuNAF488 and FSC area.

After the analysis, sort the anti-NeuNAF488-negative population in a 1.5-milliliter collection tube filled with 50 microliters of wash media using the gating strategy.