Glia-Free Cortical Neuronal Feeding Schedule - Synapse Formation
Justin T Savage, Juan Ramirez, Dolores Irala, Cagla Eroglu
Abstract
Protocol for isolating rat cortical astrocytes and producing astrocyte-conditioned media for synaptogenesis assays.
Steps
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:
| A | B |
|---|---|
| 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.
| A | B |
|---|---|
| BDNF | 20µl |
| CNTF | 20µl |
| Forskolin | 20µl |
DIV 2 - AraC feeding
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 - Media feeding
Check neurons for normal morphology under 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.
Remove 230µl of old neuronal media.
Add 250µl of fresh media.
Store in the incubator.
DIV 8 - ACM 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 AGM to the NGM for a final concentration of 50ug/ml for excitatory synapses and 100ug/ml for inhibitory synapses.
Equilibrate and warm up the NGM with ACM in the incubator (37C, 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 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 AGM to the NGM for a final concentration of 50ug/ml for excitatory synapses and 100ug/ml for inhibitory synapses.
Equilibrate and warm up the NGM with ACM in the incubator (37C, 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)
• 10ul of anti-Bassoon antibody (presynaptic marker)
• 10ul of anti-Homer1 antibody (for excitatory synapses) or anti-Gephyrin antibody (for inhibitory synapses)
• 5ul of anti-Vglu1 antibody (for excitatory synapses) or anti-VGAT antibody (for inhibitory synapses)
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)
- 10ul of secondary antibody Alexa Fluor anti-Mouse IgG2a 488
- 10ul of secondary antibody Alexa Fluor anti-Rabbit IgG1 564
- 10ul of secondary antibody Alexa Fluor anti-Guinea Pig IgG 647
- 0.5ul DAPI (1/10000)
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 2h, keeping it protected from light.
Wash 3 times with PBS.
Mount coverslips onto slides using one small drop of mounting media for each coverslip.
Seal with nail polish and dry at room temp for at least 30min before storing at 4˚C.
