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 25ml
Percoll 35ml
d H2O 40ml
Filter sterilize
60% Percoll
4X CMF-PBS 25ml
Percoll 60ml
d H2O 15ml
0.4% Trypan blue 0.3ml
Filter sterilize
Solutions:
1. CMF-PBS
NaCl 8g
KCl 0.3g
Glucose 2g
NaH2PO4.H2O 0.5g
KH2PO4 0.25g
2% stock NaHCO3 2ml
0.5ml Phenol red 0.5ml
DW up to a L
Filter and store at 4
2. Media (Add the serums and pen/strep before you use it)
BME 81.5ml
30% glucose 1.5ml
L-Glutamine 1ml
Filter and store at 4
3. Trypsin-DNase solution
DNase 50mg
Trypsin 0.5g
MgSO4.7H2O 11.25mg
CMF-PBS 49.7ml
4. DNase
DNase 50mg
BME 98.85ml
30% Glucose 1.13ml