TITLE: Glia-Free Cortical Neuron Culture
AUTHORS: Justin T Savage, Juan Ramirez, Dolores Irala, Cagla Eroglu

[DESCRIPTION]
Protocol for feeding neuronal cultures with astrocyte-conditioned media and staining them for synapses.

[STEPS]
SECTION: Preparation of Coverslips (day before prep)
5. Flame sterilize the forceps after spraying with ethanol.
SECTION: Preparation of Coverslips (day before prep)
6. Wash coverslips with ddH2O in a large petri dish (150 x 15 mm).
SECTION: Preparation of Coverslips (day before prep)
7. Set coverslips in a large petri dish (150 x 15 mm). Coat with 100ul of poly-D-lysine (PDL) solution (100ul PDL (100X) in 10ml of borate buffer).
SECTION: Preparation of Coverslips (day before prep)
8. Let the coverslips sit in PDL-B for at least 30 mins in the hood RT.
SECTION: Preparation of Coverslips (day before prep)
9. Make laminin mixture: 10 µl aliquot of mouse laminin solution + 5 ml Neurobasal media.
SECTION: Preparation of Coverslips (day before prep)
10. Add 100µl of this laminin mixture to each coverslip. Place the petri dish in a 37ᵒC incubator overnight
SECTION: Preparation of Coverslips (day before prep)
4. 
 

SECTION: Preparation of Panning Dishes (day before prep)
11. Use 10cm petri dishes for positive panning and 15cm petri dishes for negative panning. 
SECTION: Preparation of Panning Dishes (day before prep)
12. For negative plates:
To 2 15cm Petri dishes add 30 ml 50mM Tris-HCL pH9.5 + 72 µl of anti-rat IgG + IgM to one plate and 30 ml Tris-HCL pH9.5 + 72 µl of anti-mouse IgG + IgM to the other. Incubate at 4C overnight.
SECTION: Preparation of Panning Dishes (day before prep)
13. For positive plates:
To 2 10cm plates add 12 ml Tris-HCL pH9.5 + 54 µl anti-mouse IgG and IgM. Incubate at 4ᵒC.

SECTION: Cortical Neuron Isolation (Prep Day)
14. Firstly, take theL1 antibody out to thaw at RT
SECTION: Cortical Neuron Isolation (Prep Day)
15. Prepare the following solutions (enough for 9 pups)
 
 
     Tube Label      DPBS      # of Aliquots + Additive      Notes        Lo OVO      36ml      2 2ml aliquot      Add 400µl DNase before use        Hi OVO      20ml      2 2ml aliquot               BSA Buffer      76ml      2 2ml aliquot      Divide among 2 50ml Falcon Tubes        Panning  (X2 tubes)      45ml      500µl insulin + 5ml BSA buffer      Insulin, do not use if >6 weeks’ old   

 
 
SECTION: Preparation of negative panning dishes
16. Create 2 Lectin negative panning dishes: 2x 15cm Petri dishes with 30ml prep DPBS. Add 25µl Unconjugated Griffonia Simplicifolia Lectin I. Shake plate gently in a ‘+’ manner to ensure even distribution. Incubate at RT for at least 1 hour outside the hood.
SECTION: Preparation of negative panning dishes
17. Wash the mouse and rat negative panning dishes 3x with DPBS. Add 12ml BSA blocking buffer to each. Incubate for at least 1 hr at RT outside the hood.
SECTION: Preparation of negative panning dishes
18. Wash the Positive panning dish 3x with 10ml DPBS carefully. Add 5ml of mouse-anti L1 mixture. Incubate at RT until just before use outside the hood.
SECTION: Preparation of negative panning dishes
19. After the lectin has incubated for 1 hr (usually during papain digestion), wash the Lectin negative panning dishes with DPBS three times. Add 12ml of BSA buffer. Let it at RT outside the hood. This step is usually done in between the Low Ovo centrifuge step or High Ovo centrifuge step


 

SECTION: Rat brain dissection
20. Prepare dissection surface by cleaning with ethanol, placing an absorbent pad on the benchtop, and preparing tools. Will need small curved spring scissors, larger scissors for decapitation, and forceps.
SECTION: Rat brain dissection
21. Prepare 3 60mm dishes adding 5ml of DPBS to collect 6 cortices each, and one 10cm dish with 12ml of DPBS for the dissection.
SECTION: Rat brain dissection
22. Decapitate pups and de-skin the heads. Remove the skull using small scissors to trim around but above the ears up to the midline. The skull should peel off.
SECTION: Rat brain dissection
23. Remove the brains and place them into the 10cm dish with DPBS.
SECTION: Rat brain dissection
24. Using the forces remove meninges and dissect the basal ganglia, hippocampus and the cerebella. This will leave isolated cortex.
SECTION: Rat brain dissection
25. Transfer cortices to a fresh 60mm dish with DPBS.
SECTION: Rat brain dissection
26. Chop the cortices with scissors to increase the surface for enzymatic digestion.
SECTION: Tissue digestion
27. Resuspend the papain. And add the DNase.
SECTION: Tissue digestion
28. Cut off the tip of sterile plastic disposable pipette to make a larger opening.
SECTION: Tissue digestion
29. Using the plastic pipette, suck up the chopped tissue and DPBS. Tap the tip of the pipet onto the papain solution in 20 ml universal tubes without squeezing the pipet or adding liquid. Swirl gently to get a ‘tornado effect’ before letting the brain tissue settle
SECTION: Tissue digestion
30. Incubate at 32oC for 45 minutes, swirling the tissue at 15-minute intervals.
SECTION: Tissue digestion
31. Just before the end of the papain incubation, add DNase to the Lo Ovo (400ul).
SECTION: Tissue digestion
32. Once the 45-minute incubation is completed, aspirate the DPBS from the universal tubes. The tissue will stay at the bottom of the tube.
SECTION: Tissue digestion
33. Add 2 ml Lo-Ovo gently by dribbling down the side of the tube. Triturate using a 1ml pipette about 10 passes up and down. Check that there is a single cell suspension, otherwise, 1ml of Lo-Ovo can be added to help in resuspension. Add the remainder of Lo-Ovo to dissociated cells.
SECTION: Tissue digestion
34. Centrifuge the cells at 1100rpm for 11 minutes at room temperature.
SECTION: Tissue digestion
35. Aspirate off the supernatant with a vacuum without disturbing the pellet.
SECTION: Tissue digestion
36. Resuspend the cells in 2 ml Hi-Ovo. Triturate using a 1ml pipette about 10 passes. Add the remainder of Hi-Ovo.
SECTION: Tissue digestion
37. Centrifuge the cells at 1100rpm for 11 minutes at room temperature.
SECTION: Tissue digestion
38. Aspirate off the supernatant with a vacuum.
SECTION: Tissue digestion
39. Resuspend the pelleted cells in 3ml panning buffer in each tube using a P1000 pipette, up and down 10 times. Add another 7mL to bring to 10mL.
SECTION: Tissue digestion
40. Filter the cells through a Nitrex mesh (use sterile forceps to make a conical shape into a 50ml tube). Bring the volume of the filtrate to 15mL with more panning buffer. To prepare the mesh in a cone shape, place the mesh at the mouth of the tube and make a conical shape with the help of the forceps and pipette through which the tissue suspension will filter through. Use panning buffer to wet the mesh.
SECTION: Panning steps
41. Remove the DBPS/BSA from the first Lectin plate into a waste beaker. Add the filtered cells to this first Lectin plate. Let incubate at RT for 10 min.
SECTION: Panning steps
42. At the end of the incubation, shake the plate forcefully on the benchtop but keep the solution inside the plate and away from the lid. Shake such that it performs one full rotation.
SECTION: Panning steps
43. Remove the DBPS/BSA from the second Lectin plate. Dump the cell/buffer mixture into this second Lectin plate. Collect the last bit of loose cells with a P1000. Let incubate at RT for 10 min.
SECTION: Panning steps
44. At the end of the incubation, shake the plate forcefully but keep the solution inside the plate and away from the lid. Shake such that it performs one full rotation.
SECTION: Panning steps
45. Remove the BSA from the first negative panning plate. Dump the cell/buffer mixture onto this plate. Collect the last bit of loose cells with a P1000. Let incubate at RT for 15 min.
SECTION: Panning steps
46. At the end of the incubation, shake the plate forcefully but keep the solution inside the plate and away from the lid. Shake such that it performs one full rotation.
SECTION: Panning steps
47. Remove the BSA from the second negative panning plate. Dump the cell/buffer mixture onto this plate. Collect the last bit of loose cells with a P1000. Let incubate at RT for 15 min. (Just before the end of the incubation, prepare the positive panning dishes by pouring off the L1 primary solution and wash the plate 3x gently with DPBS).
SECTION: Panning steps
48. At the end of the incubation, shake the plate forcefully but keep the solution inside the plate and away from the lid. Shake such that it performs one full rotation.
SECTION: Panning steps
49. Remove the DPBS from the positive panning plates and add the cells from the second negative panning dish.
SECTION: Panning steps
50. Incubate for 45min at RT.
SECTION: Prepare 24 well plate with coverslips
51. 
 

SECTION: Prepare 24 well plate with coverslips
52. Add 500µl warmed and equilibrated NGM to each well of the 24 well plate.
SECTION: Prepare 24 well plate with coverslips
53. Pour neurobasal media onto the coverslip dish (without aspirating the existing neurobasal media on coverslips) just enough to dislodge the coverslips.
SECTION: Prepare 24 well plate with coverslips
54. Pick up with sterile forceps and put the right side up (with the PDL/lamining coating) into each well.
SECTION: Neuronal collection from positive panning
55. Using a 5ml serological pipette set to slow, gently remove the media (and loose unstuck cells). Wash at least 5 times with 5ml of Panning buffer by tilting the dish slightly towards self so that DPBS pools at the marked triangle. Gently place the dish back so the buffer covers the entire dish. Keep the 5th wash on the plate and check under a microscope until it looks clean from debris.
SECTION: Neuronal collection from positive panning
56. Remove the last wash of the panning buffer and add 5ml of panning buffer to collect the neurons. Use a P1000 pipette to carefully pipet up and down all over the surface of a dish. Collect the neurons in a 50ml tube. Add another 5ml of panning buffer, and check under the microscope to make sure all the neurons were collected. Place the cells in a 50ml conical tube and centrifuge at 1100rpm for 11 min at RT.
SECTION: Neuronal collection from positive panning
57. Resuspend the neuronal pellet in 2ml of warmed and equilibrated NGM.
SECTION: Neuronal collection from positive panning
58. Count the cells using a hemocytometer or an automated cell counter.
SECTION: Neuronal collection from positive panning
59. Plate the cells onto their coverslips at a density of 70,000/well. Store in the incubator at 10% CO2
SECTION: Prepare Neuronal Growth Media (NGM)
1. This recipe makes 20ml of neuronal media. Make fresh per use.

To a 50ml tube, add the following media components:
 
     Reagent       Volume        Neurobasal      19ml          Pen/strep   (100x)      200µl                                  Glutamine   (100x)                           200µl          Sodium   Pyruvate (100x)      200µl          B27   (50x)                                      400µl        
 
SECTION: Prepare Neuronal Growth Media (NGM)
2. Sterile filter these components through a syringe filter. Place media in the incubator with thecap unscrewed. Allow the media to warm and equilibrate for at least 45 minutes. (Media will have an orange color and bubbles)
SECTION: Prepare Neuronal Growth Media (NGM)
3. Add growth factors right before the time to use the media.

 
     BDNF       20µl                                    CNTF       20µl         Forskalin       20µl