Mar 25, 2026
Version 1
Human Brain Nuclei Isolation Using Sucrose Gradient Ultracentrifugation V.1
- Zhang Lab1
- 1Yale University
Protocol Citation: Zhang Lab 2026. Human Brain Nuclei Isolation Using Sucrose Gradient Ultracentrifugation. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9yrm4g3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2022
Last Modified: March 25, 2026
Protocol Integer ID: 59970
Keywords: Human brain Nuclei, Sucrose gradient, Isolation, Ultracentrifugation, ASAPCRN, isolation of human brain nuclei, human brain nuclei isolation, using sucrose gradient ultracentrifugation, sucrose gradient ultracentrifugation this protocol, human brain nuclei, gradient ultracentrifugation, isolation
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000529
Abstract
This protocol describes about isolation of human brain nuclei by sucrose gradient ultracentrifugation.
Attachments
392-854.docx
20KB
Materials
Reagents
MilliQ water DNase-, RNase-free water
| A | B | |
| Sucrose Homogenization Buffer | ||
| Sucrose | 2 M | |
| Hepes (pH 7.9) | 10 mM | |
| KCl | 25 mM | |
| EDTA (pH 8.0) | 1 mM | |
| Glycerol | 10% | |
| Ribonuclease (RNase) inhibitors freshly added | 80 U/ml | |
| A | B | |
| Nuclei Suspension Buffer | ||
| Hepes (pH 7.4) | 15 mM | |
| NaCl | 15 mM | |
| KCl | 60 mM | |
| MgCl2 | 2 mM | |
| CaCl2 | 3 mM | |
| RNase inhibitors freshly added | 80 U/ml) | |
Materials
- Eppendorf tube
- surgical blade
- Ultracentrifuge
- SW28 rotor
- haemocytometer slide (C-Chip, DHCN015)
38.5 mL Open-Top Thinwall Ultra-Clear Tube 25 x 89mm - 50PkBeckman CoulterCatalog #344058
Troubleshooting
Isolation of Human Brain Nuclei
1h 10m
Take brain tissue sample out of -80 °C freezer and put on dry ice. Transfer the sample from the bag or tube to a petri dish and carefully cut a small piece off with a surgical blade, keeping it on dry ice. Replace the remaining piece in the bag and put the small piece in a labeled Eppendorf tube. Keep the Eppendorf tube on dry ice. Repeat for all samples, using a different petri dish and surgical blade for a new sample.
Optional: Weigh Eppendorf tubes if needed and record weights. 50 mg per sample.
Pour 25 mL per sample of sucrose homogenization buffer into 50 mL Falcon tube and place On ice . Add 1 µL of RNase Inhibitor (40 U/μL ) per mL of homogenization buffer. Shake tube to mix.
Pipette 15 mL homogenization buffer with RNase Inhibitor into 15 mL Dounce tissue grinder On ice . Add tissue piece and homogenize.
Note
Note: Slowly break big tissues (first 3-4 strokes up and down), then dounce up and down 10 times with the “loose” pestle followed by 10 times with the “tight” pestle.
Note: Pull the pestle out slowly to avoid spilling. Each sample takes 5-10 minutes.
Label bottom of 50-mL ultracentrifuge tube (in case it breaks in the centrifuge). Pipette 10 mL fresh homogenization buffer to the tube as a cushion, and carefully pour the 15 mL of homogenate on top of it to maintain two separate layers.
Place the ultracentrifuge tubes in the swing buckets. Weigh and balance the buckets containing the samples by adding a few drops of fresh homogenization buffer to the top.
Ultracentrifuge at 25000 rpm, 4°C, 01:00:00 in SW28 rotor for ultracentrifuge.
Note
- Do not use a fixed angle rotor. Always use a swinging bucket.
- Thin wall 50-mL tube: BECKMAN 344058 (Tube, Thinwall, Ultra-Clear™)
- Wash glassware: Rinse each piece with tap water 10 times. Then rinse with MilliQ water 5 times. Lay out to dry on paper towels. Once dry, rinse one time with DNase-, RNase-free water, air dry and store for next time.
1h
Add 2 µL RNase Inhibitor (40 U/μL ) per mL of nuclei suspension buffer, making 5 mL total buffer per sample. Mix and place On ice .
Remove all the supernatant by pouring off into a petri dish. Place upside-down in petri dish for a few seconds. Cut off the wall of the thin centrifuge tube with a clean razor blade.
Resuspend the nuclei pellet at the bottom of the cut ultracentrifuge tube in 500 µL nuclei suspension buffer. Pipette gently up and down in the solution 10–20 times. Transfer this buffer into a labeled Eppendorf tube and repeat with an additional 500 µL nuclei suspension buffer. Place On ice .
In a new labeled Eppendorf tube, make a 1:1 dilution of the nuclei suspension and Trypan Blue. Count nuclei (round blue dots) using haemocytometer slide (C-Chip, DHCN015). Count each 4x4 grid at the corners and average the counts.
Concentration of the nuclei: Average count x 2 (dilution in Trypan Blue) x 104/mL.
Spin down nuclei at 800 x g, 4°C and 9 acceleration/deceleration for 00:10:00 .
10m
Carefully remove supernatant with pipette.
Note
Do not disturb the pellet at the bottom.
Resuspend in nuclei suspension buffer: the ideal concentration for 10X is 1000 nuclei/μL.
Make a new 1:1 dilution of the nuclei suspension and Trypan Blue. Recount nuclei to ensure the concentration is adequate for 10x (700-1200 nuclei/μL). Ideally, there would be 50 nuclei per 4x4 grid in the haemocytometer slide.
Submit samples for 10x Genomics Chromium loading (single cell 3’ V3.1 chemistry).