Granular cells culturing.
Coating the dishes:
Sterilize the dishes, coverslips or grids (in the glow discharger) and coat
them with 1:50 dilution of matrigel in CMF-PBS immediately after.
Leave for 15 min minimum at RT.
Rinse 3X with CMF-PBS right before you are ready to plate your cells.
Percoll Gradient
Load 5ml of 60% percoll on the bottom of a 15ml falcon tube.
Then add 5ml of 35% percoll on top.
Leave it in the hood (you can leave it there until you are ready to use it)
Dissection:
Dissect cerebellum in cold CMF-PBS
Peel off meninges
Move the cerebellum in a fresh falcon tube.
Add 1ml of warm (370) Trypsin-DNase in CMF-PBS
Incubate at 37 for 5 min
Remove Trypsin_DNase carefully but leave a little.
Add 1ml DNase (370)(without Trypsin)
Triturate with a extra fine Pasteur pipet 10 times
Spin at 2000rpm for 5 min at RT , brake ON
Remove sup and resuspend the pellet in 1ml CMF-PBS and 0.25ml DNase
using a fine Pasteur pipet.
Load 1.25ml on the top of the percoll gradient SLOWLY
Spin at 3000rpm for 30min at RT, NO BRAKE (it takes 40min)
Harvest:
Remove the second band from the top and put it in a 15ml falcon tube.
Fill it up to 15ml with CMF-PBS cold
Spin at 2000 rpm 5 min at 40
Aspirate the sup.
Plating:
Add 0.1ml DNase and 0.5ml of BMB +10% Horse serum +5% Fetal bovine
serum+1% sterp/pen. MAKE FRESH EVERY TIME.
Transfer the cells to an eppendorf.
Spin for 5min at 4.5 speed.
Resuspend pellet in 0.5 medium.
Count cells
Plate up to 3X106 (1X106)
Plate, leave for ½ hr in the incubator and then add some more media.
Leave for 4 days before you change the media.
GRADIENT:
35% Percoll
4X CMF-PBS
Percoll
d H2O
Filter sterilize
25ml
35ml
40ml
60% Percoll
4X CMF-PBS
Percoll
d H2O
0.4% Trypan blue
Filter sterilize
25ml
60ml
15ml
0.3ml
Solutions:
1. CMF-PBS
NaCl
KCl
Glucose
NaH2PO4.H2O
KH2PO4
8g
0.3g
2g
0.5g
0.25g
2% stock NaHCO3
0.5ml Phenol red
DW
Filter and store at 4
2ml
0.5ml
up to a L
2. Media (Add the serums and pen/strep before you use it)
BME
30% glucose
L-Glutamine
Filter and store at 4
81.5ml
1.5ml
1ml
3. Trypsin-DNase solution
DNase
Trypsin
MgSO4.7H2O
CMF-PBS
50mg
0.5g
11.25mg
49.7ml
4. DNase
DNase
BME
30% Glucose
50mg
98.85ml
1.13ml