TITLE: Glia-Free Cortical Neuronal Feeding Schedule - Synapse Formation
AUTHORS: Justin T Savage, Juan Ramirez, Dolores Irala, Cagla Eroglu

[DESCRIPTION]
Protocol for isolating rat cortical astrocytes and producing astrocyte-conditioned media for synaptogenesis assays.

[STEPS]
SECTION: Neuronal growth media (NGM) for feeding
1. This recipe makes 20ml of neuronal media. Make fresh per use
SECTION: Neuronal growth media (NGM) for feeding
2. 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        
 
SECTION: Neuronal growth media (NGM) for feeding
3. 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)
SECTION: Neuronal growth media (NGM) for feeding
4. Add growth factors right before the time to use the media.
 
     BDNF       20µl                                    CNTF       20µl         Forskolin       20µl    
 
SECTION: DIV 2 - AraC feeding
5. 
 

SECTION: DIV 2 - AraC feeding
6. Check neurons for normal morphology under a microscope.
SECTION: DIV 2 - AraC feeding
7. 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.
SECTION: DIV 2 - AraC feeding
8. Remove 230µl of old neuronal media.
SECTION: DIV 2 - AraC feeding
9. Add 250µl of fresh media.
SECTION: DIV 2 - AraC feeding
10. Store in the incubator
SECTION: DIV 3 - remove AraC from media
11. Check neurons for normal morphology under a microscope.
SECTION: DIV 3 - remove AraC from media
12. 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.
SECTION: DIV 3 - remove AraC from media
13. Remove 230µl of old neuronal media.
SECTION: DIV 3 - remove AraC from media
14. Add 250µl of fresh media.
SECTION: DIV 3 - remove AraC from media
15. Store in the incubator.
SECTION: DIV 6 - Media feeding
16. Check neurons for normal morphology under microscope.
SECTION: DIV 6 - Media feeding
17. 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.
SECTION: DIV 6 - Media feeding
18. Remove 230µl of old neuronal media.
SECTION: DIV 6 - Media feeding
19. Add 250µl of fresh media.
SECTION: DIV 6 - Media feeding
20. Store in the incubator.
SECTION: DIV 8 - ACM feeding
21. Check neurons for normal morphology under a microscope.
SECTION: DIV 8 - ACM feeding
22. 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.
SECTION: DIV 8 - ACM feeding
23. Place the NGM on ice to cool down.
SECTION: DIV 8 - ACM feeding
24. Thaw the ACM on ice.
SECTION: DIV 8 - ACM feeding
25. Add AGM to the NGM for a final concentration of 50ug/ml for excitatory synapses and 100ug/ml for inhibitory synapses.
SECTION: DIV 8 - ACM feeding
26. Equilibrate and warm up the NGM with ACM in the incubator (37C, 10% CO2) before adding it to the cells.
SECTION: DIV 8 - ACM feeding
27. Remove 230µl of old neuronal media.
SECTION: DIV 8 - ACM feeding
28. Add 250µl of fresh media.
SECTION: DIV 8 - ACM feeding
29. Store in the incubator.
SECTION: DIV 11 - ACM feeding
30. Check neurons for normal morphology under a microscope.
SECTION: DIV 11 - ACM feeding
31. 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.
SECTION: DIV 11 - ACM feeding
32. Place the NGM on ice to cool down.
SECTION: DIV 11 - ACM feeding
33. Thaw the ACM on ice.
SECTION: DIV 11 - ACM feeding
34. Add AGM to the NGM for a final concentration of 50ug/ml for excitatory synapses and 100ug/ml for inhibitory synapses.
SECTION: DIV 11 - ACM feeding
35. Equilibrate and warm up the NGM with ACM in the incubator (37C, 10% CO2) before adding it to the cells.
SECTION: DIV 11 - ACM feeding
36. Remove 230µl of old neuronal media.
SECTION: DIV 11 - ACM feeding
37. Add 250µl of fresh media.
SECTION: DIV 11 - ACM feeding
38. Store in the incubator.
SECTION: DIV 13 - Neuron culture fixation and staining
39. 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)      
SECTION: DIV 13 - Neuron culture fixation and staining
40. In a chemical fume hood, aspirate media from cells and immediately add 500µl of warm 4% PFA to each well.
SECTION: DIV 13 - Neuron culture fixation and staining
41. Fix the neurons for 7 minutes at room temperature.
SECTION: DIV 13 - Neuron culture fixation and staining
42. Wash 3 times with PBS.
SECTION: DIV 13 - Neuron culture fixation and staining
43. Add 200ul of blocking solution to each well and block for 30min at room temperature.
SECTION: DIV 13 - Neuron culture fixation and staining
44. Remove the blocking solution and add 200µl of primary antibody solution to each well.  Incubate at 4˚C overnight.
SECTION: DIV 14 - Secondary antibody staining and mounting
45. 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)
SECTION: DIV 14 - Secondary antibody staining and mounting
46. Remove the primary antibody and wash 3 times with PBS
SECTION: DIV 14 - Secondary antibody staining and mounting
47. Add 200µl of secondary antibody solution to each well and incubate at room temperature for 2h, keeping it protected from light.
SECTION: DIV 14 - Secondary antibody staining and mounting
48. Wash 3 times with PBS.
SECTION: DIV 14 - Secondary antibody staining and mounting
49. Mount coverslips onto slides using one small drop of mounting media for each coverslip.
SECTION: DIV 14 - Secondary antibody staining and mounting
50. Seal with nail polish and dry at room temp for at least 30min before storing at 4˚C.