Gradient Centrifugation Based Purification of Nuclei: A Technique to Obtain Ultrapure Fraction of Intact Nuclei Using Density Gradient Centrifugation

Add 200 microliters of 50% iodixanol solution to the nuclei for a final concentration of 25% iodixanol and mix 10 times with the pipette set to 300 microliters.

Then, add 300 microliters of 29% iodixanol solution under the 25% mixture using a P1000 fine tip to avoid mixing the layers. Add 300 microliters of 35% iodixanol solution under the 29% mixture with a P1000 fine tip. Then, slowly remove it.

Place the samples in a swinging bucket centrifuge and spin them for 20 minutes at 3,500 x g at 4 degrees Celsius with the brake off.

Gently remove the samples without shaking and observe them under light. A clear white band of 95% pure nuclei should be visible between the second and third layer.

To isolate the nuclei, aspirate the top layers to expose the white nuclei band at the interface. Collect the nuclei band in a 200-milliliter volume and transfer it to a fresh tube. Then, filter it with a 20-micrometer filter.

Check the nuclei under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. They should be round, and the nuclear membrane should not be distorted.