Mar 30, 2023
Single cell dissociation of brain organoids
- 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Protocol Citation: michela.deleidi , Maria Jose Perez J., Hariam Raji 2023. Single cell dissociation of brain organoids. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwj799lmk/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, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79538
Keywords: brain organoids, cell dissociation, Papain Dissociation System, ASAPCRN, single cell dissociation of brain organoid, brain organoid, single cell dissociation, protocol details about single cell dissociation, cell dissociation, cell, brain
Abstract
This protocol details about single cell dissociation of brain organoids.
Attachments
405-879.docx
15KB
Materials
Kit:
Papain Dissociation System. Papain Dissociation SystemWorthington Biochemical CorporationCatalog #LK003150
Reconstitute powders.
- Add 5 mL Earle´s medium into Papain Vial (1 Vial/2 organoids).
- Add 500 µL Earles´s medium into DNAse vial.
- Add 35 mL Earle´s medium into Inhibitor vial (1 vial/10 organoids).
Single cell dissociation of brain organoids
27m
Mix 500 µL DNAse with 5 mL Papain.
Note
Note: MIX GENTLY.
Transfer single or pooled organoid to 60 mm dish.
Aspirate excess media, add 2.5 mL Papain + DNAse solution.
With a razor blade mince organoid (<1 mm).
Transfer plate to an orbital shaker 70 rpm, 00:30:00 (inside incubator).
With 1-mL pipette dissociate pieces (Mix up-down 30 times).
Put in orbital shaker 00:20:00 .
20m
In the meantime, add 5 mL Earle´s medium + 3 mL Inhibitor to a 15-mL conical tube.
Remove samples from the orbital shaker. With a 1-mL tip, mix up-down 30 times.
Take 2 mL (upper part) into new tube using a 40 µm cell strainer. Wait 1-3 min to debris to settle.
Transfer cell suspension to the inhibitor tube. Invert to mix 5 times.
Centrifuge 300 rpm, Room temperature, 00:07:00 .
7m
Aspirate supernatant, resuspend in 500 µL to 1 mL 0.5% BSA-PBS (Up-down 30 times).
Filter the resuspended cells (900 µL ) with a 30 µm cell strainer.
Count the cells for the final suspension and dilute. Resuspend at 1000 cells/μl in 0.04% BSA-PBS.