Dec 01, 2025

Public workspaceSingle Cell Dissociation of Cultured Neurons

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l29574v1y/v1
  • Marc Bosse1,
  • Joerg Isensee2,
  • Tim Hucho2,
  • Sean Bendall1,
  • Kausalia Vijayaragavan1
  • 1Stanford University;
  • 2Department of Anesthesiology and Intensive Care Medicine, Experimental Anesthesiology and Pain Research, University Hospital of Cologne, Cologne, Germany
Kausalia Vijayaragavan
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l29574v1y/v1
Protocol Citation: Marc Bosse, Joerg Isensee, Tim Hucho, Sean Bendall, Kausalia Vijayaragavan 2025. Single Cell Dissociation of Cultured Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l29574v1y/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: October 30, 2024
Last Modified: December 01, 2025
Protocol Integer ID: 111291
Keywords: single cell dissociation of cultured neuron, single neural cells from monolayer culture, single neural cell, cultured neuron, cell neuron dissociation, single cell dissociation, individual neurons in isolation, individual neuron, cell culture, crucial technique in cell culture, monolayer culture, cell, insights into cellular mechanism, neurodevelopmental process, cellular mechanism, macs enrichment, use in macs enrichment
Abstract
Single-cell neuron dissociation is a crucial technique in cell culture that allows scientists to study individual neurons in isolation, providing insights into cellular mechanisms, gene expression, and neurodevelopmental processes. Here, we describe a method to obtain single neural cells from monolayer culture for use in MACS enrichment, FACS sorting, and re-culture.
Materials
Collagenase B 10x (4.0 U/mL). Sigma-Aldrich 11088807001
Collagenase P 10x (50.0 U/mL). Sigma-Aldrich l11213857001
Cell Disassociation Buffer Thermo Fisher Scientific 13151014
N2B27 Medium Gibco
Pen/Strep
CloneR2. StemCell Technologies 100-0691
BSA 30%, cell culture grade. Sigma cat # A9576
Benzonase Nuclease, Benzonase Nuclease HC, 90%, Size=25 ku
Thermo Scientific 202-1SPK with 7.9 mm wide tip
Thermo Scientific 202-1SPK 

Troubleshooting
Material
Collagenase B 10x (4.0 U/mL). Sigma-Aldrich 11088807001
Collagenase P 10x (50.0 U/mL). Sigma-Aldrich l11213857001
Cell Disassociation Buffer Thermo Fisher Scientific 13151014
N2B27 Medium Gibco
Pen/Strep
CloneR2. StemCell Technologies 100-0691
BSA 30%, cell culture grade. Sigma cat # A9576
Benzonase Nuclease, Benzonase Nuclease HC, 90%, Size=25 ku
Thermo Scientific 202-1SPK with 7.9 mm wide tip
Method
Preparation of Collagenase B Stock Solution (10x)
  1. Collagenase B comes in a bottle containing 100 mg of lyophilized powder. The label indicates the amount of units/mg. (first bottle was 0.38 u/mg, i.e. ≈ 40 units).
  2. Calculate the total amount of units in that bottle and hydrate its content with N2B27 Medium to make a stock solution of 4.0 units/ml.
  3. Filter and aliquot 1 ml of the stock solution in 15 ml centrifuge tubes.
  4. Store at Temperature-20 °C .
  5. This solution is not sterile.

Preparation of Collagenase P Stock Solution (10x)
  1. Collagenase P comes in a bottle containing 100mg of lyophilized powder. The label indicates the amount of units/mg.
  2. Calculate the total amount of units in that bottle and hydrate its content with N2B27 Medium to make a stock solution of 50 units/ml.
  3. Aliquot 500 ul of the stock solution in 1 ml appendorf tubes.
  4. Store at Temperature-20 °C .
  5. This solution is not sterile.

Preparation of Collagenase B (1x at 0.4U/ml) and P (1x at 5U/ml) Digestion Buffers:
  1. Dilute at respective Collagenase stock solutions to 1x in N2B27 medium.
  2. Prepare volume accordingly to get 3 mL per T25.

ABCDE
Reagentsstock []working dilutionworking []vol (ul)
Benzonase25ku/ml 1:100000.00251.5
BSA30% 1:300.99%100
CloneR210x 1:101x300
Collagenase B40units/ml1:1000.4unit/ml30
Penicillin-Streptomycin (10,000 U/mL)100x 1:100130
1x N2B272307
final volume (ul)3000
Collagenase B Digestion Buffer.

ABCDE
Reagentsstock []working dilutionworking []vol (ul)
Benzonase25ku/ml 1:100000.00251.5
BSA30% 1:300.99%100
CloneR210x 1:101x300
Collagenase P50units/ml1:105units/ml300
Penicillin-Streptomycin (10,000 U/mL)100x 1:100130
1x N2B271767
final volume (ul)3000
Collagenase P Digestion Buffer.

Prepare clean up medium
  1. Add 2 mL of 30% BSA to 2 mL of N2B27 to make 15% BSA. Keep these on ice until use.
Prepare post sort medium
  1. Add 0.5 mL of BSA (30%) to 15 mL of N2B27 (final 1% BSA).
  2. Add Clone R2.
  3. Add GF specific for cell type of interest (NGF for neurons etc) as supplement to promote survival.
Prepare sort tube
  1. Add 3 mL of sort medium per 15 mL falcon tube.
  2. Roll the tube horizontally to coat the entire side wall (this very important to prevent loss of cells to the side of the tube.
Cell Disassociation (GENTLE IS THE RULE)
2h 37m
  1. Remove culture medium
  2. Add freshly made Collagenase B Digestion Buffer, 3 mL/ T25.
  3. Incubate Duration01:00:00 at Temperature37 °C in the incubator .
  4. Add freshly made Collagenase P Digestion Buffer to Collagenase B Digestion Buffer/Cell suspension, 3 mL/ T25.
  5. Incubate Duration01:00:00 at Temperature37 °C in the incubator .
  6. Collect cell suspension in 15 mL tube with large caliber transfer pipette (7.9 mm).
  7. Centrifuge Duration00:05:00 at 400g.
  8. Add 1 mL of Cell Dissociation buffer.
  9. Suspend cell pellet by flicking the tube..
  10. Incubate Duration00:10:00 at Temperature37 °C water bath .
  11. Add 4 mL of ice cold sort medium (N2B27 + 1 % BSA + 1x Clone R2).
  12. Incubate on TemperatureOn ice ice for Duration00:15:00 .
  13. Triturate gently by aspirating up and down with a large caliber transfer pipette.
  14. Pipette up and down slowly with a 1 mL pipette.
  15. Centrifuge Duration00:05:00 at 400g pre-cooled to Temperature4 °C .
  16. Suspend by flicking the tube in 1 mL of sorting medium and reserve on ice.
Note
Axon stumps and disrupted cells are removed by adding ice cold 15% BSA gradient centrifugation. 

  1. Add 3 mL of ice cold clean up medium to a new 15 tube.
  2. Gently layer the cell suspension on top of the clean up medium.
  3. Centrifuge Duration00:08:00 at 120g at Temperature4 °C .
  4. Remove the supernatant carefully and keep aside.
Note
Axon stumps and disrupted cells are mostly floating in the supernatant. Viable cells are in the pellet.

  1. Check supernatant for viable cells and recovery.
  2. Add 2 mL of sort medium to the pellet. Re-suspend pellet by flicking the tube. DO NOT PIPETTE
  3. Pass cell through cell strainer (70 µM).
  4. Perform cell counts and adjust concentration accordingly for sorting.
  • Ideally for good sort low cell density at 1 million per ml maximum.


2h 43m
Protocol references
PKA-RII subunit phosphorylation precedes activation by cAMP and regulates activity termination.
Jörg Isensee, Melanie Kaufholz, Matthias J Knape, Jan Hasenauer, Hanna Hammerich , Humberto Gonczarowska-Jorge , René P Zahedi , Frank Schwede , Friedrich W Herberg , Tim Hucho 
J Cell Biol. 2018 Jun 4;217(6):2167–2184. doi: 10.1083/jcb.201708053. PMID: 29615473

Nociceptive properties of in vitro differentiated neurons derived from human pluripotent stem cells.
Marc Bossé, Joerg Isensee, Sakthikumar Mathivanan, Isabel Devesa Giner, Ruth Iceta, Tim Hucho, Antonio Ferrer-Montiel, Sean Bendall and Kausalia Vijayaragavan.
Unpublished. 2024