Apr 20, 2026
Glia-Free Cortical Neuronal Feeding Schedule - Neuronal Morphology
- Justin T Savage1,
- Juan Ramirez1,
- Dolores Irala2,
- Cagla Eroglu1,2,3,
- Sarah van Veen4,5
- 1Department of Neurobiology, Duke University Medical Center, Durham, NC 27710;
- 2Department of Cell Biology, Duke University Medical Center, Durham, NC 27710;
- 3Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710;
- 4KU Leuven;
- 5Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Justin T Savage, Juan Ramirez, Dolores Irala, Cagla Eroglu, Sarah van Veen 2026. Glia-Free Cortical Neuronal Feeding Schedule - Neuronal Morphology. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv52bw4v1b/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 10, 2025
Last Modified: April 26, 2026
Protocol Integer ID: 118077
Keywords: free cortical neuronal feeding schedule, treatment of cortical neuron, cortical neuron, neuronal morphology this protocol, astrocyte, neuronal morphology, glia, conditioned medium, dendrite length, spermidine, subsequent analysis of dendrite length
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
Abstract
This protocol outlines the treatment of cortical neurons with astrocyte-conditioned medium (ACM) or spermidine and subsequent analysis of dendrite length.
Materials
Penicillin-Streptomycin (10,000 U/mL) (Gibco, 15140148)
Sodium Pyruvate (GIBCO 11360)
Neurobasal plus (Gibco, A35029-01)
B27 plus (Gibco, A35828-01)
Forskolin (5 mM; 1000X)
Add 12ml DMSO to 50 mg of Forskolin (Sigma, F6886).
Make 20 ul aliquots; store -20˚C.
BDNF – (Peprotech, 450-02). Master stock should be prepared at 1mg/ml in 0.2%BSA DPBS and is stored at -80˚C as 100ul aliquots.
Preparation of 1000x Working Stocks
Thaw on ice one master stock aliquot (100ul) of BDNF. Cool down on ice 2ml of filtered 0.2% BSA (Sigma A-8806) in DPBS solution. Add BDNF master stock aliquot to the BSA solution. Mix well but gently to avoiding foaming. Aliquot in 0.5ml tubes 20ul each, concentration 50ug/ml (1000X). Flash freeze the aliquots in liquid nitrogen and store at -80C. Working concentration: 50ng/ml, final concentration on neurons 25ng/ml.
CNTF – (Peprotech, 450-13). Master stock should be prepared at 1mg/ml in 0.2%BSA DPBS and is stored at -80˚C as 50ul aliquots.
Preparation of 1000x Working Stocks
Thaw on ice one master stock aliquot (100ul) of CNTF. Cool down on ice 2.5ml of filtered 0.2% BSA (Sigma A-8806) in DPBS solution. Add CNTF master stock aliquot to the BSA solution. Mix well but gently to avoiding foaming. Aliquot in 0.5ml tubes 20ul each, concentration 20ug/ml (1000X). Flash freeze the aliquots in liquid nitrogen and store at -80C. Working concentration: 20ng/ml, final concentration on neurons 10ng/ml.
Spermidine (Sigma, Cat# S2626)
Master stock prepared at 500 mM in 0.1 M MOPS (pH 7, adjusted with KOH) and stored at -80°C as aliquots.
Dilute in NGM to obtain an intermediate working stock. Mix well. Final concentration on neurons: 10 nM.
Lipofectamine‱ 2000 Transfection Reagent (Invitrogen, Cat# 11668027)
Opti-MEM (GIBCO, Cat# 11058021)
Antibody Blocking Buffer - 150 mM NaCl, 50 mM Tris-Base, 1% BSA, 100 mM L-lysine.
In a 1L baker add 4.383g NaCl, 3.025g Tris-Base (Fisher, Cat. No: BP152-5), 5g BSA (Sigma, Cat. No:A2153) and 9.125g L-Lysine monohydrochloride (Sigma, Cat. No: L-1137). Add 350-400ml of ddH2O and mix well. Once mixed, adjust pH with HCl to 7.5. Finally, add 5ml of Sodium Azide (NaN3). Add ddH2O to final volume 500ml. Filter through 0.22μm filter and store 4C.
VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Cat# H-1200-10).
PFA 16% (Electron Microscopy Sciences, 19210)
Normal Goat Serum (NGS) - (ThermoFisher 01-6201)
Triton X-100 (Thermo Fisher, Cat# 28314)
Anti-GFP antibody (Aves Labs, Cat# GFP-1020, RRID:AB_10000240)
Goat anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor 488 (Thermo Fisher, Cat# A-11039, RRID:AB_2534096)
Safety warnings
- Maintain sterility and avoid contamination during all steps.
- Follow institutional guidelines for the disposal of biological and chemical waste.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Neuronal growth media (NGM) for feeding
This recipe makes 20ml of neuronal media. Make fresh per use
- To a 50ml tube, add the following media components:
| Reagent | Volume | |
| Neurobasal plus | 19ml | |
| Pen/strep (100x) | 200µl | |
| Sodium Pyruvate (100x) | 200µl | |
| B27 plus (50x) | 400µl |
Sterile filter these components through a syringe filter. Place media in the incubator with cap unscrewed. Allow the media to warm and equilibrate for at least 45 minutes. (Media will have an orange color and bubbles)
Add growth factors right before the time to use the media.
| BDNF | 20µl | |
| CNTF | 20µl | |
| Forskolin | 20µl |
DIV 2 - AraC feeding
Note
Neurons are extremely sensitive to environmental changes, so only half of the media is replaced.
Check neurons for normal morphology under a microscope.
Prepare neuronal media with Neurobasal plus + B27 plus according to recipe fresh + add AraC (1:10000). Let equilibrate for at least 45 min in the incubator with the cap unscrewed.
Remove 230µl of old neuronal media.
Add 250µl of fresh media.
Store in the incubator
DIV 3 - remove AraC from media
Check neurons for normal morphology under a microscope.
Prepare neuronal media with Neurobasal plus + B27 plus according to recipe fresh. Let equilibrate for at least 45 min in the incubator with the cap unscrewed.
Remove 230µl of old neuronal media.
Add 250µl of fresh media.
Store in the incubator.
DIV 6 - Neuron transfection
2h 30m
Check neurons for normal morphology under microscope.
Transfect neurons with GFP plasmid, using Lipofectamine 2000.
Prepare Lipofectamine solution (per well): 100 μl Opti-MEM + 2 μl Lipofectamine. Pipet together and incubate for 00:05:00 at Room temperature .
5m
Prepare DNA-Lipofectamine mixture: 100 μl Opti-MEM + 2 μl Lipofectamine + 2 μl of 0.5 μg/μl GFP plasmid. Pipet together and incubate for 00:05:00 at Room temperature .
5m
Add the Lipofectamine solution (prepared in ) to the DNA-Lipofectamine mixture (prepared in ), mix and incubate for 00:20:00 at Room temperature .
20m
Remove 300 μl of medium from neurons and save in sterile tube. Add 200 μl of transfection mixture to neurons. Incubate for 02:00:00 at 37 °C .
2h
Remove all medium from neurons. Add 50% saved medium and 50% new medium. Return neurons to incubator.
DIV 8 - ACM/spermidine feeding
Check neurons for normal morphology under a microscope.
Prepare neuronal media with Neurobasal plus + B27 according to recipe fresh. Let equilibrate for at least 45 min in the incubator with the cap unscrewed.
Place the NGM on ice to cool down.
Thaw the ACM on ice.
Add the following to NGM depending on experimental condition:
| Condition | Additive | Final concentration | |
| ACM | ACM | 100 ug/ml | |
| Spermidine | Spermidine | 10 nM | |
| Control | Vehicle | equivalent volume |
Equilibrate and warm up the NGM with additive in the incubator (37 °C , 10% CO2) before adding it to the cells.
Remove 230µl of old neuronal media.
Add 250µl of fresh media.
Store in the incubator.
DIV 11 - ACM/spermidine feeding
Check neurons for normal morphology under a microscope.
Prepare neuronal media with Neurobasal plus + B27 according to recipe fresh. Let equilibrate for at least 45 min in the incubator with the cap unscrewed.
Place the NGM on ice to cool down.
Thaw the ACM on ice.
Add the following to NGM depending on experimental condition:
| A | B | C | |
| ACM | ACM | 100 ug/ml | |
| Spermidine | Spermidine | 10 nM | |
| Control | Vehicle | equivalent volume |
Equilibrate and warm up the NGM with additive in the incubator (37 °C , 10% CO2) before adding it to the cells.
Remove 230µl of old neuronal media.
Add 250µl of fresh media.
Store in the incubator.
DIV 13 - Neuron culture fixation and staining
The following solutions should be prepared:
a) 4% PFA in PBS – In a 50ml conical tube add one 16% PFA ampule (10ml), 26ml of ddH2O and 4ml of 10X PBS. Mix and place at 37˚C to warm up. For storing keep away from the light at 4C.
b) Blocking and permeabilization solution (0.2% Triton) - This recipe makes 5ml of blocking solution, sufficient for 1 24-well plate.
• 2.4ml of antibody-blocking buffer
• 2.5ml of Normal Goat Serum (NGS)
• 100ul of 10% Triton.
c) Primary antibodies solution: This recipe makes for 5ml, sufficient for 1 24-well plate.
• 4500µl Antibody blocking buffer
• 500µl Normal Goat Serum (NGS)
• 5ul of anti-GFP antibody
In a chemical fume hood, aspirate media from cells and immediately add 500µl of warm 4% PFA to each well.
Fix the neurons for 7 minutes at room temperature.
Wash 3 times with PBS.
Add 200ul of blocking solution to each well and block for 30min at room temperature.
Remove the blocking solution and add 200µl of primary antibody solution to each well. Incubate at 4˚C overnight.
DIV 14 - Secondary antibody staining and mounting
a) Secondary antibodies solution: This recipe makes for 5ml, sufficient for 1 24-well plate.
- 4500µl Antibody blocking buffer
- 500µl Normal Goat Serum (NGS)
- 5ul of anti-Chicken IgY (H+L) Secondary Antibody, Alexa Fluor 488
Remove the primary antibody and wash 3 times with PBS
Add 200µl of secondary antibody solution to each well and incubate at room temperature for 3h, keeping it protected from light.
Wash 3 times with PBS.
Mount coverslips onto slides using one small drop of VECTASHIELD Antifade Mounting Medium with DAPI.
Seal with nail polish and dry at room temp for at least 30min before storing at 4˚C.
Imaging
Acquire images using a fluorescence microscope (e.g. Keyence BZ-X800) at 20x magnification.
Analysis
Manually analyze dendrite length using the NeuronJ plugin within the FIJI software.
Protocol references
Irala D, Wang S, Sakers K, Nagendren L, Ulloa Severino FP, Bindu DS, et al. Astrocyte-secreted neurocan controls inhibitory synapse formation and function. Neuron. 2024;112(10):1657-75 e10.