Mar 19, 2026
Protocol for Dissociation and Culture of Midbrain Astrocytes and Neurons
- Elena Coccia1
- 1Icahn School of Medicine at Mount Sinai
Protocol Citation: Elena Coccia 2026. Protocol for Dissociation and Culture of Midbrain Astrocytes and Neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vze28xvx1/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: February 12, 2026
Last Modified: March 19, 2026
Protocol Integer ID: 243126
Keywords: midbrain astrocytes, neurons, cell dissociation, culture, CNTF, culture of midbrain astrocyte, midbrain astrocyte, astrocyte, neurons this protocol, protocol for dissociation, neuron, dissociation
Abstract
This protocol aims to dissociate midbrain astrocytes and neurons, culture them in a defined ratio, and maintain their viability and differentiation potential for subsequent experiments.
Materials
TrypLE Select (Cell dissociation reagent)
Accutase (Cell dissociation reagent)
Geltrex (Growth factor reduced matrix)
Neuronal media (Stage-appropriate neuronal differentiation media)
CNTF (Ciliary neurotrophic factor)
Black 96-well plate (Plastic-bottom plate (Greiner))
Reagents:
Cell Dissociation TrypLE Select - Enzyme for cell dissociation
Cell Dissociation Accutase - Enzyme for cell dissociation
Matrix Geltrex - Extracellular matrix for cell culture
Growth Factor CNTF - Promotes neuronal survival and differentiation
Troubleshooting
Problem
Low cell viability after dissociation
Solution
Ensure optimal incubation times and avoid excessive pipetting.
Problem
Poor attachment of cells to the plate
Solution
Ensure Geltrex is properly prepared and polymerized before adding media.
Safety warnings
Use personal protective equipment (PPE) including gloves and lab coats. Handle all reagents according to safety data sheets (SDS).
Cell Dissociation
Dissociate midbrain astrocytes and neurons using TrypLE Select and Accutase.
Dissociation of Astrocytes
1. Add TrypLE Select to the midbrain astrocyte culture. 2. Incubate at 37 °C for 3 minutes. 3. Gently pipette up and down to dissociate cells. 4. Neutralize with 10 mL of complete medium. 5. Centrifuge at 300 g for 5 minutes at room temperature. Count cells.
Dissociation of Neurons
1. Add of Accutase to the neuron culture. 2. Incubate at RT for 3 minutes. 3. Gently pipette up and down to dissociate cells. 4. Neutralize with 10 mL of complete medium. 5. Centrifuge at 300 g for 5 minutes at room temperature. 6. Count cells.
Cell Seeding
Pool the cells and plate into a 96-well plate.
Cell Pooling and Seeding
1. Combine the resuspended astrocytes and neurons at a 5:1 ratio (100,000 neurons to 20,000 astrocytes). 2. Mix gently and spin down again. 3. Remove supernatant and resuspend into Geltrex supplemented with 10µM of Y-27632, 10ng/ml CNTF, 20ng/ml BDNF, 20ng/ml GFNF. Aliquot 5 µL droplets into each well of the black 96-well plate. 3. Allow droplets to polymerize at 37°C for 10 minutes. 4. Fill each well with 100 µL of neuronal differentiation media supplemented with 10 ng/mL CNTF.