TITLE: Astrocyte Isolation and ACM Production
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: Cortical Astrocyte Isolation (Prep Day)
1. 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: Rat brain dissection
6. 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
7. 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
8. 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
9. Remove the brains and place them into the 10cm dish with DPBS.
SECTION: Rat brain dissection
10. Using the forces remove meninges and dissect the basal ganglia, hippocampus and the cerebella. This will leave isolated cortex.
SECTION: Rat brain dissection
11. Transfer cortices to a fresh 60mm dish with DPBS.
SECTION: Rat brain dissection
12. Chop the cortices with scissors to increase the surface for enzymatic digestion.
SECTION: Tissue digestion
13. Resuspend the papain. And add the DNase.
SECTION: Tissue digestion
14. Cut off the tip of sterile plastic disposable pipette to make a larger opening.
SECTION: Tissue digestion
15. 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
16. Incubate at 32oC for 45 minutes, swirling the tissue at 15-minute intervals.
SECTION: Tissue digestion
17. Just before the end of the papain incubation, add DNase to the Lo Ovo (400ul).
SECTION: Tissue digestion
18. 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
19. 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
20. Centrifuge the cells at 1100rpm for 11 minutes at room temperature.
SECTION: Tissue digestion
21. Aspirate off the supernatant with a vacuum without disturbing the pellet.
SECTION: Tissue digestion
22. 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
23. Centrifuge the cells at 1100rpm for 11 minutes at room temperature.
SECTION: Tissue digestion
24. Aspirate off the supernatant with a vacuum.
SECTION: Cortical Astrocyte Isolation (Prep Day)
2. Prepare Astrocyte Growth Media (AGM)
This recipe makes 500ml of filtered astrocyte growth media. Store at 4C.
Combine and filter the following reagents:
DMEM 450ml
FBS 50ml
Penstrep (100x) - 5ml
Glutamate (100x) - 5ml 
Sodium Pyruvate (100x) - 5ml
Insulin - 5ml
N-Acetyl Cysteine - 500µl
Hydrocortisone - 500µl
SECTION: Preparation of culture flasks
3. Dilute 100µl of PDL in 10ml water.
SECTION: Preparation of culture flasks
4. Coat 75cm2 culture flasks with 10 µg/ml PDL for at least 30 mins at room temperature.
SECTION: Preparation of culture flasks
5. Wash 3 times with sterile ddH2O, then add 15ml AGM to each flask. Let sit in the incubator with the lid slightly twisted off during prep to let the CO2 levels and temperature equilibrate.
SECTION: Tissue digestion
25. Resuspend the cells in 2-3ml warmed AGM. Bring up to 10ml. Filter the cell mixture through 30m Nitex membrane 1 mL at a time into a 50ml Falcon tube. Remember to Ethanol/flame sterilize forceps.
SECTION: Tissue digestion
26. Centrifuge the filtered cells at 1100rpm for 10 minutes at room temperature.
SECTION: Tissue digestion
27. Aspirate off the supernatant and resuspend the cells in 2 mL AGM.
SECTION: Tissue digestion
28. Bring up the volume to 10ml to count the cells.
SECTION: Tissue digestion
29. Plate cells at very high density: 15 million cells per 75cm2 culture flask as a minimum. Better if can be between 17-20 millon. Add the required amount to the prepped flasks.
SECTION: Tissue digestion
30. Store in incubator with cap slightly unscrewed.
SECTION: DIV 3 Astrocyte Shake-off
31. Warm-up AGM and DPBS in a water bath
SECTION: DIV 3 Astrocyte Shake-off
32. Check astrocytes for normal morphology under a microscope
SECTION: DIV 3 Astrocyte Shake-off
33. Wash the cells with 10ml DPBS at least 3 times
SECTION: DIV 3 Astrocyte Shake-off
34. Add 15ml of DPBS and shake the flasks at least 10 times (this allows for other cell types to detach from the coating).
SECTION: DIV 3 Astrocyte Shake-off
35. Aspirate the DPBS and dislodged cells
SECTION: DIV 3 Astrocyte Shake-off
36. Add another 15ml of DPBS. Check under a microscope to see if cell debris and other cells have been dislodged. There should be patches of ‘holes’ into which the astrocytes can grow into. If insufficient, shake more/harder.
SECTION: DIV 3 Astrocyte Shake-off
37. Add 15ml of AGM
SECTION: DIV 3 Astrocyte Shake-off
38. Store in incubator
SECTION: DIV 5 Feed Astrocytes with AraC
39. Warm-up AGM in a water bath and add AraC (1:10000)
SECTION: DIV 5 Feed Astrocytes with AraC
40. Check astrocytes for normal morphology under a microscope
SECTION: DIV 5 Feed Astrocytes with AraC
41. Remove old AGM and replace with fresh AGM+AraC (15ml/flask)
SECTION: DIV 5 Feed Astrocytes with AraC
42. Store in the incubator
SECTION: DIV 7 Astrocyte Passage
43. 
 


SECTION: DIV 7 Astrocyte Passage
44. Warm-up trypsin, AGM and DPBS in a water bath
SECTION: DIV 7 Astrocyte Passage
45. Dilute 100µl of PDL in 10ml water and coat a 10 cm culture dish with 10ml of 10 µg/ml PDL for at least 30 mins at room temperature.
SECTION: DIV 7 Astrocyte Passage
46. Wash 3 times with sterile ddH2O, then add 10ml AGM to the plate. Let sit in the incubator during the astrocyte passaging to let the CO2 levels and temperature equilibrate.
SECTION: DIV 7 Astrocyte Passage
47. Remove old AGM media in the flask
SECTION: DIV 7 Astrocyte Passage
48. Rinse the flask with PBS without magnesium and calcium 3 times
SECTION: DIV 7 Astrocyte Passage
49. Add 10 ml pre-warmed trypsin to each flask. Incubate for 5 minutes at 37˚C in the incubator, lying flat (not stacked) to have even heat.
SECTION: DIV 7 Astrocyte Passage
50. Tap flask to loosen astrocytes and collect with 10ml pipette all the astrocytes that are now are dislodged.
SECTION: DIV 7 Astrocyte Passage
51. Centrifuge at 1.1 x 1000RPM for 10mins to pellet the astrocytes
SECTION: DIV 7 Astrocyte Passage
52. Vacuum off the supernatant
SECTION: DIV 7 Astrocyte Passage
53. Resuspend the pellet in 2ml of AGM. Pipette up and swirl to dislodge the pellet. Dispense and swirl gently until a cell suspension is achieved.
SECTION: DIV 7 Astrocyte Passage
54. Plate the astrocytes in the 10cm dish at 3 million as a minimum. Better if can be between 4-5 million.
SECTION: DIV 7 Astrocyte Passage
55. Store in the incubator.
SECTION: Astrocyte Conditioning Medium
56. Allow freshly plated cells to grow up to 4 days in AGM.
SECTION: Astrocyte Conditioning Medium
57. Wash plates with DPBS 2~3 times.
SECTION: Astrocyte Conditioning Medium
58. Add 10 mL serum-free conditioning media (see materials), and incubate without disturbing (4~5 days).
SECTION: Astrocyte Conditioning Medium
59. Prepare a centrifugal concentrator by washing it with distilled water by centrifuge ~8 mins @ 4000g @ 4C.
SECTION: Astrocyte Conditioning Medium
60. Pour off H2O from centrifugal concentrator
SECTION: Astrocyte Conditioning Medium
61. Collect ACM into 50 mL tubes, and centrifuge (1100g, 5 mins) to pellet cell debris.
SECTION: Astrocyte Conditioning Medium
62. Carefully transfer ACM from 50 mL tubes to centrifugal concentrator w/o disturbing pellet.
SECTION: Astrocyte Conditioning Medium
63. Spin centrifugal concentrator for ~60 mins @ 4000 g 4C (or down to around 1 mL, 20X).
SECTION: Astrocyte Conditioning Medium
64. Prepare sterile low protein binding tubes for aliquots.
SECTION: Astrocyte Conditioning Medium
65. Make ~100ul aliquots for experiments plus one extra aliquot for measuring protein concentration, freeze with liquid N2, and keep at -80 until use.