TITLE: Primary hippocampal and cortical neuronal culture and transfection
AUTHORS: Ryan J. Farrell, Alexandros C Kokotos, Timothy A. Ryan

[DESCRIPTION]
Neurons consist some of the most complex cells. The culture of primary neurons from murine brains has advanced the understanding of neuronal function at the cellular and molecular level. This protocol presents an established method to isolate and culture hippocampal and cortical neurons from rat and mouse brains along with a method to sparsely transfect these neurons. 

[BEFORE_START]
On average, 1 rat pup (2 hippocampi) yields approximately 10 dishes of hippocampal neurons.
Plating media and MEM should be warmed before use in the water bath (37 oC) and filter-sterilized before addition to cells.
Dissection tools should be kept in sporicidin.
Dissection time should be about 5 minutes per pup. 
Pipette solutions slowly on the side of the tubes to prevent bubbles.
Incubators should be set to 5% CO2 37 oC.

[GUIDELINES]
The animals should be used in accordance with protocols approved by national and institutional regulatory organizations.

[STEPS]
SECTION: Preparing Dishes, 1 Day before
69. 
SECTION: Preparing dishes, 1 Day before
1. For each dish, place a coverslip in a p35.
SECTION: Preparing dishes, 1 Day before
2. Take an 100X poly-L-ornithine aliquot and dilute in 10 mL ultrapure water (final conc. 10000 µg/µL).

SECTION: Preparing dishes, 1 Day before
3. Add ~ 200 µL of poly-L-ornithine to the center of each coverslip.

SECTION: Preparing dishes, 1 Day before
4. Place in incubator  .

SECTION: Preparing for culture
5. Add 8 mL of HBSS + 20 % (v/v) FBS to a dissecting dish.

SECTION: Preparing for culture
6. Add 4 mL of HBSS + 20 % (v/v) FBS to a p35.

SECTION: Preparing for culture
7. Place in fridge until ready to dissect.
SECTION: Preparing for culture
8. Prepare dissection hood and fill a bucket with ice.
SECTION: Preparing for culture
9. Turn on centrifuge and cool to 4 °C.

SECTION: Preparing for culture
10. Collect rat pups from your animal facility. 
You should collect one pup for every 10 dishes. Pups should be P0 – P2.
SECTION: Dissection (Hippocampal) also see video
11. Perform the dissection on a piece of cold marble. Marble is kept in the -20 °C. Change out the marble every two pups.

SECTION: Dissection (Hippocampal) also see video
12. Decapitate rat pup using the scissors. Place rat carcass in a biohazard bag and dispose of according to your institution’s policy.
SECTION: Dissection (Hippocampal) also see video
13. Using tweezers stab through the eyes and use the sharp blade (#11) to cut through the skull.
SECTION: Dissection (Hippocampal) also see video
14. While keeping tweezers in the eyes, using another pair of tweezers, remove skull.
SECTION: Dissection (Hippocampal) also see video
15. Use the scoop to remove the brain and place in a dissecting dish filled with cold HBSS + 20 % (v/v) FBS.

SECTION: Dissection (Hippocampal) also see video
16. Cut the brain in half using the rounded scalpel blade (#10).
SECTION: Dissection (Hippocampal) also see video
17. Stab into the cerebellum and roll the cortex away using the micro knife.
SECTION: Dissection (Hippocampal) also see video
18. Flip over and isolate the hippocampus using the micro knife.
SECTION: Dissection (Hippocampal) also see video
19. Remove meninges with tweezers making sure to remove meninges in the groove between the subiculum and the hippocampus.
SECTION: Dissection (Hippocampal) also see video
20. Stab into the subiculum and use the micro knife the remove the dentate gyrus.
SECTION: Dissection (Hippocampal) also see video
21. Flip over and remove the hippocampus following the dark crescent moon shape.
SECTION: Dissection (Hippocampal) also see video
22. Place in the p35. Store on ice until all hippocampi are dissected.
SECTION: Dissection (Hippocampal) also see video
23. Once all hippocampi are dissected cut each hippocampus into 4 pieces.
SECTION: Culture
24. Move the dissected hippocampi in HBSS + 20 % (v/v) FBS to a 15 mL falcon tube.

SECTION: Culture
25. Aspirate off the dissection media.
SECTION: Culture
26. Wash with 8 mL of HBSS + 20 % (v/v) FBS.

SECTION: Culture
27. Wash 3 times with 8 mL HBSS.

SECTION: Culture
28. Add 7 µL DNase to 5 mL of pre-aliquoted digestion solution and dissolve the 40 mg trypsin aliquot.

SECTION: Culture
29. Remove HBSS from hippocampi and filter the digestion solution onto the hippocampi.
SECTION: Culture
30. Leave the 15 mL tube at an angle to allow for optimal digestion. Digest for 5 min.
(Note: If the sample starts to stick together add more DNase).
SECTION: Culture
31. After 5 min, aspirate the digestion solution.

SECTION: Culture
32. Wash 2 times with 8 mL of HBSS + 20 % (v/v) FBS.

SECTION: Culture
33. Wash 3 times with 8 mL HBSS.

SECTION: Culture
34. Add 7 µL DNase to 5 mL of pre-aliquoted dissociation solution. Filter on to hippocampi

SECTION: Culture
35. Using a P1000 pipettor, slowly pipette the cells up and down until fully dissociated. Make sure to not create bubbles.
SECTION: Culture
36. Place a cell strainer in a 50 mL centrifuge tube and pour the cell suspension over the strainer.
SECTION: Culture
37. Wash the 15 mL tube that contained the cell suspension with 6 mL of HBSS + 20 % (v/v) FBS. Add the solution using a serological pipette to the strainer.

SECTION: Culture
38. Take the strained cell suspension and place in a new 15 mL conical and spin at 300 x g 4 °C for 10 min.

SECTION: Culture
39. During the spin aspirate off the poly-o from the dishes and wash two times with 0.5 mL ultrapure water.

SECTION: Culture
40. After the spin is complete, remove the media and wash with 6 mL of HBSS. Resuspend the pellet and spin again at 300 x g 4 °C for 10 min.
SECTION: Culture
41. During this spin, add the cloning cylinders.  Using heated forceps, place the cylinder in the grease and then place the cylinder in the center of the coverslip.
SECTION: Culture
42. After this spin, aspirate off the media and resuspend in plating media. Our rule of thumb is to resuspend in 0.5 mL of plating media per pup dissected.
When aspirating media after pelleting cells, you can increase yield by removing the final mL of media with the P1000.
43. Then add 10 µL to the hemocytometer and count the cells. You want ~38,000 cells/cylinder. Each cylinder holds 100 µL.

44. Dilute cells with plating media to the appropriate concentration.
45. Add 100 µL of plating media with cells per cylinder.

46. Add 3 mL of MEM to the outside of each dish. 

SECTION: Feeding the cells
48. Feeding should be done DIV2.
Replace 60 µL of plating media in the cylinder with 60 µL of warm filtered feeding media.

SECTION: Transfection
49. Transfection should be done between DIV6 – DIV7.
Warm MEM and Feeding Media in the water bath.
SECTION: Transfection
50. Turn on and place heating blanket in the hood.
SECTION: Transfection
51. For each dish triple rinse by removing 60 µL media and replacing with 60 µL of warm MEM three times.

SECTION: Transfection
52. Replace the MEM outside the cylinder with 3 mL of feeding media.

SECTION: Transfection
53. Return cells to the incubator for 30 min.

SECTION: Transfection
54. Prepare DNA mix as below, but do not add HEBS. Wait at least 10 min.

SECTION: Transfection
55. Remove cells from the incubator.
SECTION: Transfection
56. Add HEBS, while vortexing, to the DNA mix.  Immediately add 12 µL of DNA mix to each dish. Pipette quickly and do not add dropwise.

SECTION: Transfection
57. Return cells to the incubator for 12 min.  Do not open the incubator during this time.

SECTION: Transfection
58. After 12 min, remove cells from the incubator and remove 60 µL of media.

SECTION: Transfection
59. Break off the cylinder to flood with Feeding Media.
SECTION: Transfection
60. Return to incubator until ready to image.
SECTION: DNA Mixes (HeBS)
61. These are starting suggestions for your transfection.  Optimize as needed. These recipes are good for seven dishes, scale as appropriate. Plasmids for transfection should be from a Midi or Maxi Prep.
SECTION: DNA Mixes (HeBS)
62. Single Transfection: 6 µg of plasmid, 4.5 µL CaCl2, Ultrapure water to 45 µL, 45 µL HEBS

SECTION: DNA Mixes (HeBS)
63. Double Transfection: 4 µg of one plasmid, 5 µg of the other plasmid (Add 5 µg of the plasmid that gives a lower transfection yield), 4.5 µL CaCl2, Ultrapure water to 45 µL, 45 µL HEBS

SECTION: DNA Mixes (HeBS)
64. Triple Transfection: 3 µg of each plasmid, 4.5 µL CaCl2, Ultrapure water to 45 µL, 45 µL HEBS

SECTION: 10.	Additional notes
65. This culture protocol can also be used as is for postnatal mouse cultures. The only difference is that feeding the cells should be done later on, typically 4-5 days in-vitro.
SECTION: 10.	Additional notes
66. This culture protocol can be used for postnatal cortical cultures, with the following differences and notes. During dissection, still remove the hippocampus (and olfactory bulb if present).
Make sure to remove the meninges. 
Do not use the cell strainer. 
Half rat cortex should yield approximately 10 million cells. 
Do not do more than one full brain per tube. 
Double the amount of DNase. 
If you have trouble dissociating leave longer with the trypsin. 
Feed cells later once the glia appear more confluent typically 4-5 days in-vitro.
47. Place cells in the incubator.