1.Protocol for preparing hippocampal neuronal cultures
flowchart
1.ordering animals
2.preparation of media
3.preparation of culture plates
4.dissection of the hippocampus
5.culture of cells
1.order of animals
rat : timed pregnant Wister rat (E15-E20) with about 15 fetuses
mouse: timed pregnant mouse (E15-W20) with about 10 fetuses
2.preparation of media
DMIT (for culture)
1000ml
DMEM 2 (Nissui) glutamate(-) 9.5g
NaHCO3 (wako) 2.4g
Hepes (pH7.4) (dojin) 20mM (20mMstock 20ml)
Glucose (Dexitorose) 3.5g
Penicilin & Streptomycin (Behlinger) 2ml
Transferrin(Sigma)* 100mg
Putrescine(Sigma)* 16mg
Insulin(Wako,gamma)* 5mg (10mg/mlstock,500ｵl)
Progesteron(Sigma)* 20nM (40ｵM stock 500ｵl)
Na2SeO3(Sigma,s5261)* 30nM (20ｵg/mlstock 260ｵl)
*If you make DMIT(-),these factors are not included.
NaOH pH 7.5
filter through (0.22ｵm)
sol-P(for dissection)
200ml
NaCl 120mM 5M 4.8ml
KCl 5mM 1M 1ml
Glucose 25mM 900mg
Pipes/NaOH (pH7.0) 20mM 1M 4ml
CaCl2* 1mM 1M 200ｵl
MgCl2* 1mM 1M 200ｵl
*If you make f-Sol-P, these factors are not included.
filter through (0.22ｵm)
Trypsin solution (×10) (for dissection)
trypsin (Gibco) 10mg/ml 200mg
DNase(Behringer) 5mg/ml 100mg
dissolve into f-SolP solution 20ml
Trypsin inhibitor solution(×10) (for dissection)
trypsin inhibitor(Gibco) 3mg/ml 60mg
DNase(Behringer) 0.4mg/ml 8mg
dissolve into SolP solution 20ml
coating buffer
polyethylene imine 0.2vol%
or poly-L-lysin(Sigma P-2636) 1mg/ml
dissolve into borate buffer 0.1M pH8.5
filter through (0.22ｵm)
3.preparation of culture plates
3-1 preparation of plastic plates
all steps must be performed in a laminar flow food!
1. cover each wells with coating solution and leave in room temparature overnight
2. rinse each wells with stelile water, 2 times
note: these plates are desireble to be prepared just before dissection of hippocampus
3-2 preparation of coverslips for imaging
It is so difficult for neurons to adhere on glass coverslips. Coating coverslips is very clitical to prepare a good condition culture.
1. place coverslips in staining lack(Hirasawa) and rinse in water
2. dip into concentrated HNO3 for 18 to 36hours and clean them
3. rinse coverslips with sterile water 2 times for 1hour and wipe the surface of glass
4. tap off excess water and dry in oven or autoclave
5. sterilize flexiperm discs (Hereaous) in autoclave
coverslip(Matsunami)
flexiperm disc(Heraeus)
following steps must be performed in a laminar flow food!
6. put on the burner
7. dry up excess water of coverslips
8. press flexiperm disc on coverslip in 10cm dish not to make a space between them
9. cover each wells with coating solution and leave in room temparature overnight
10. rinse each wells with stelile water, 2 times
note: these plates are desireble to be prepared just before dissection of hippocampus
4.dissection of the hippocampus
check all materials are ready?
comprete all procedures within 2 hours!
1. euthanize pregnant rat or mouse with diethylether in a desiccator
2. cut skin and remove uterus en bloc
3. place in a ethanol-wiped dish and take fetuses out
4. decapitate fetuses, dissect out the brains cutting along the saggital, frontal and sigma sutures and place them in f-SolP
5. strip away the meninges and dissect hippocampi
6. place hemisphere of brain and hippocampi in f-SolP media
7. place them in a 15-ml centrifuge tube, and bring the total volume to 4.5ml with f- SolP
8. add 0.5ml of trypsin (10fold) and incubate at 37C for 5min
9. spin down cells for 20sec centrifuge and remove supernatant
10. add trypsin inhibitor and dissociate cells by pipetting up and down, first in a normal Pasteur pipette, then in a pipette whose tip has been fire polished to a small diameter
11. continue pipetting until no lumps of tissue remain
12. spin down cells for 4 mins at 1000rpm
13. decant sup and tap well
14. add culture medium and count cell density
15. place in coated dish with your favorable density
ex. high density 2.5E5 / well (24well) very low density 0.5E4 / well (24well)
16. replace culture medium with fresh one, once or twice a week
5.culture of cells
note:do not dry the cell surface in medium change!
1. replace culture medium with fresh DMIT/HS10% after 2 or 3 days
2. replace culture medium with fresh DMIT/BSA0.1% after 1 week
3. to reduce glial proliferation, add AraC 5.0E-6M after 2 or 3 days