Encyclopedia of Experiments: Cancer Research
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Isolation of Nuclei from Fresh Frozen Glioma Tissues - ConceptTranscript
Begin by transferring 10 to 60 milligrams of fresh frozen tissue sample to a prechilled Petri dish. Mince or chop the fresh frozen tissue with a razor blade into small pieces on ice. Add 500 microliters of chilled nuclei lysis buffer onto the tissue in the Petri dish.
Then, transfer the mixture to a Douncer. Dounce the tissue pieces with the "loose" pestle for about 20 strokes until friction is reduced. Then, dounce it with the 'tight' pestle for 20 strokes to achieve complete tissue homogenization.
Transfer the homogenate into a prechilled 2-milliliter tube and add 1 milliliter of chilled lysis buffer into the Douncer, rinse it, and add it to the tube. Mix it gently and incubate it on ice for 5 minutes, mixing with a wide-bore pipette tip one to two times during the incubation.
Filter the entire homogenate using a 30-micrometer strainer mesh and collect it into a 15-milliliter Falcon tube. Then, transfer it back into a new prechilled 2-milliliter tube. A single strainer is typically sufficient for the entire homogenate.
Check the sample under a light microscope to verify the removal of large debris and the intactness of the nuclear membrane. Nuclei need not be round and the nuclear membrane should not be distorted. If debris is present, repeat the filtration.
Next, centrifuge the nuclei on a benchtop centrifuge for 5 minutes. Remove the supernatant, leaving behind approximately 50 microliters of the nuclei-containing pellet. Gently resuspend the pellet in another milliliter of nuclei lysis buffer and incubate it for 5 minutes on ice.
Repeat the centrifugation. Then, remove the supernatant without disturbing the pellet. Add 500 milliliters of HB and incubate the sample for 5 minutes without resuspending. Then, resuspend the nuclei in another 1 milliliter of HB.
Centrifuge for another 5 minutes. Then, remove the supernatant. Resuspend nuclei in 200 microlitres of HB and transfer the suspension into a new 2-milliliter tube.
