Oct 01, 2025
Irradiation and single-cell dissociation of hESCs and cortical spheroids
- Annika Martin1,
- Hanqin Li1,2,
- Dirk Hockemeyer1,2
- 1University of California, Berkeley, Berkeley, CA 94720, USA;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 2081, USA
Protocol Citation: Annika Martin, Hanqin Li, Dirk Hockemeyer 2025. Irradiation and single-cell dissociation of hESCs and cortical spheroids. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xwbzlqe/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: January 11, 2024
Last Modified: October 01, 2025
Protocol Integer ID: 93400
Keywords: ASAPCRN, cortical spheroids this protocol, cell dissociation of hesc, cortical spheroid, cortical spheroids for multi, procedure of irradiation, cell dissociation, irradiation, sequencing protocol, hesc, sequencing
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000486
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-024409
Abstract
This protocol describes the procedure of irradiation and single-cell dissociation of hESCs and cortical spheroids for MULTI-Seq barcoding and sequencing.
Protocol Overview
A. Irradiation of hESC
B. hESCs derived cortical spheroids
Note
A list of reagents and relevant vendor information can be found in the table listed under the materials tab.
Attachments
Materials
Reagents
| Item | Vendor | Catalog Number | |
| 10x HBSS (Ca and Mg Free) | Invitrogen | 14185-052 | |
| Sodium Pyruvate 100mM | Life Tech | 11360070 | |
| D-Glucose | Sigma | G8769-100ml | |
| HEPES pH 7.3 | Invitrogen | 15630-080 | |
| Y-27632 – ROCK Inhibitor | Chemdea | CD0141 | |
| Accutase | Thermo Fisher Scientific | SCR005 | |
| Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) | Sigma | E6635-100G | |
| 2-Mercaptoethanol | Sigma | M3148 | |
| L-Cystine solution | Sigma-Aldrich | C7352-100G | |
| Papain suspension | Worthington Biochemical | LS003126 | |
| PBS- (without Ca and Mg) | Corning | MT21031CV | |
| PBS+ (with Ca and Mg) | Thermo Fisher | 14040-182 | |
| Trypsin Inhibitor | Sigma | T9253-5G | |
| Costar‱ 6-well Clear Flat Bottom Ultra-Low Attachment 6-well plate | Corning | 3471 |
Irradiation of hESCs
10m
3 days before irradiation, passage cells and let them grow to approximately 50% confluence.
On the day of irradiation, change media to hESC media + 10uM Rock Inhibitor (RI).
Using a discrete cesium source, irradiate cells in plates at desired dosage. We used 0, 0.5, 2, 5 and 10 Gy.
24 hours post irradiation, dissociate cells by aspirating media and adding 1 mL of accutase per well of a 6-well plate. Return to incubator for 00:05:00 to 10 min.
5m
Resuspend cells using 10 mL of PBS- to dilute accutase. Spin down for 00:05:00 at 300 x g .
5m
Aspirate supernatant and resuspend cells in 1 mL PBS- per well.
Label cells with MULTIseq oligos as described in MULTI-Seq Barcoding and Library Preparation protocol (dx.doi.org/10.17504/protocols.io.kxygx3xzkg8j/v1) and proceed with sequencing.
hESCs Derived Cortical Spheroids
2h
Note
Dissociation Media (500mL):
48.9 mL 10x HBSS (Ca and Mg Free)
440 mL H2O
5 mL Sodium Pyruvate 100mM
1.1 mL D-Glucose
5 mL HEPES pH 7.3
Differentiate hESCs to Cortical spheroids as described in Cortical Spheroid Differentiation protocol (https://doi.org/10.17504/protocols.io.5jyl8po57g2w/v1.).
At 25 days in differentiation, two organoids per condition were transferred with 1.5 mL of media into a 1.5mL Cryovial with screw top. Using a discrete cesium source, irradiate organoids in vials at desired dosage. We used 0, 0.5, 2, 5 and 10 Gy.
- Post-irradiation, transfer both organoids per condition into one well of a 6-well low adherence plate with 2.5 mL additional media. Add Rock Inhibitor (RI) to final concentration of 10uM.
24 hours post irradiation, prepare 5 mL of Activated Papain Solution per irradiation condition:
To each 5mL of Dissociation Media, add 12 µL of 0.5M EDTA, 23ul of 0.1uM β-mercaptoethanol, 5 µL of 5.5mM L-Cystine solution, and 172 µL of papain suspension.
Transfer the solution to the 37 °C water bath for ~00:20:00 to 30 minutes to activate. The solution will gradually go from cloudy to transparent.
20m
Sterilize by passing the solution through a 0.22 μm syringe filter.
Collect cortical spheroids and sediment. Aspirate the media and rinse once with PBS+.
Add 5 mL Activated Papain Solution to each well and return to the incubator for ~ 00:45:00 .
45m
While dissociating, prepare 15mL Trypsin Inhibitor solution per 5 mL of Activated Papain Solution.
For each 15mL Trypsin Inhibitor Solution: Add 15 mg Trypsin Inhibitor to 15 mL of usual organoid media. Mix thoroughly and sterilize by filtering through a 0.22 μm syringe filter.
After 00:45:00 of dissociation, add 5 mL of Trypsin Inhibitor solution to each well and very gently triturate with a 5mL strippette to dissociate. Avoid excessive pipetting and bubbles.
45m
Strain mixture through a 70μm cell strainer into a 50mL conical tube.
Use remaining 10 mL Trypsin Inhibitor Solution to rinse the well to collect any cells left behind, then pass this through the same 70μm strainer to release any cells still stuck in the mesh.
Spin down cells at 300 x g for 00:05:00 and aspirate the media.
5m
Resuspend dissociated cells in 4 mL 0.2% BSA in PBS+ with RI per condition, strain through a 40um mesh cell strainer and FACS sort for desired number of live cells and to remove debris.
Spin down live cells at 300 x g for 00:05:00 . Resuspend in 0.2% BSA in PBS+ with 10uM RI in appropriate volume according to single-cell sequencing protocol, count cells, and proceed to single-cell sequencing according to manufacturer protocol.
5m