Culture of spinal cord neurons of mouse embryos
1. Preparaion
-Cold hank’s (3dishes/mouse)
-Cold D1SGH (3dishes/4 mouse)
-Ice pack
-Syringe
Ice Pack
2.Isolate spinal cord from embryo
-Wash connected embryo with hank’s using syringe and put on the ice pack.
-Wash connected embryo with hank’s again.
-Detach each of embyos as follows; left hand hold tweezer and right hand hold sharp
tweezer and put in the glucose solution.
c- A
B
V
C
V
Ice Pack
-Move spinal cord in A and B to C of D1SGH solution
2. Isolation of spinal cord from embryo with microscope.
Lid
Gauze
2. Chop spinal cord isolated
3. First, up and down 15ml of just MEM media in 50ml conical tube with pipet and add
chopped cell.
4. Centrifuge at 1,000rpm for 5 min and suction with pipet.
5. Add 15ml of papain solution and put in incubator 37oC, 30min.
-Papain solution is composed of 2ml of papain (BM) and 28ml of glucose solution.
6. Add 15 ml of MEM containing 10% FCS/10% HS and centrifuge at 2,000rpm for 8
min. and suction until it remains about 5ml.
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Dr. Lee’s Lab
7. Make solution as follows;
-Gentamycin
500l
-DNAse
500l (Stock soln. 4mg/ml in PBS)
-MEM(10% FCS/10%HS) 50ml
8. Add 20ml of solution to cell pellet and up and down 30 times and stand for 10mins. at
r.t. and move supernatant to 75cm2 flask.
9. Repeat No. 8 again and make to 100ml with MEM containing 10% FCS/10% HS.
That is, concentration of cells become about 106 cells/ml
10. Plate 2ml to 60mm dish. (usually plate 50 dishes of 60mm dish with 4 mouse)
11. Cuture of spinal cord neurons of mice-embryo.
weeks
1
2
3
4
day
Wed
Thu
Mon
Fri
Mon
Fri
Mon
Isoate of Exchange
Decant
Decant Decant Decant Experimeembryo’ with 3ml of 1ml
of 1ml of 1ml of 1ml of nt
s spinal NH2 media
media and media media media
cord
then add 1 and
and
and
neurons NH2 media
ml
of then
then
then
Neurobasal
FDU-U
add 1 add 1 add 1
medium
containing ml of ml of ml of
(Final 500 ml) NH2
NH2
NH2
NH2
+
media.
media. media. media.
N2 supplement (NH2
(Gibco#0252) 38ml
5 ml
+
+
FDU-U
L-Gln.
solution
1.25ml
2 ml)
+
Fungizone
5 ml
+
Gentamycine
(Gibco)
5ml
Coating Buffer
1. Weigh out 1.185 g of borax and 0.775 g of boric acid and add to a 500ml beaker.
2. Obtain 250 ml of water and add to reagents.
3. Stir until mixed well.
4. Add ~30 ml of mixture to 50 ml conical tube.
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5. Obtain a pipette and blue tip with the end slightly cut off.
6. Add 400 l of polyethyleneimine to conical tube and mix thoroughly with vortex.
7. Add to mixture and continue to stir.
8. Adjust pH to 8.4 and then filter into 500 ml bottle.
Coating Dishes
Borate Buffer (borax 1.185g, boric acid 0.775g in 250 ml H2O (pH 8.4)
 Add 400 l polyethyleneimine polymer solution (Fluka 03880) to 250 ml buffer
solution
-Note : Very thick. Add with cut pipette tip into tube with 10 ml of buffer ;
Vortex, mix together.
 To each glass bottom dish add  1.5 ml of buffer.
 Leave dishes (covered) in hood; allow buffer to dry for  2hrs.
 Wash and aspirate with sterile H2O 3X
 Leave dishes open in hood for at least 1/2 hr (not necessary)
 Add medium (MEM-10% FBS), store in incubator.
D1SGH solution
1.
2.
3.
4.
5.
6.
7.
8.
Measure out 50 ml of sucrose/glucose stock solution.
Add to 1000ml beaker.
Measure out 50 ml of D1 saline stock solution.
Add to beaker.
Measure out 28 ml of Hepes stock solution.
Add to beaker.
Measure 872 ml of UPH2O.
Add to beaker.
9. Place stir bar in beaker and mix well.
10 Obtain two 500 ml bottles and a top bottle filter (.45m) and filter solution into both
bottles.
20X D1 Saline
1. Weigh out;
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a) 40.0 g of NaCl
b) 2.0 g of KCl
c) 0.18 g of NaH2PO4
d) 0.15 g of KH2PO4
2. Obtain 200 ml of UPH2O. Place in volumetric flask.
3. Add salts. Stir with stir bar. Bring volume to 250 ml.
4. Filter solution into sterile bottles.
HEPES solution
1.
2.
3.
4.
5.
Weigh out 16.76 g of HEPES.
Place in volumetric flask and add 160 ml of UPH2O.
Dissolve and adjust pH to 7.4.
Adjust volume to 250 ml.
Filter solution into sterile bottles.
Sucrose/Glucose solution
1. Weigh out 15.0 g of glucose.
2.
3.
4.
5.
Weigh out 37.5 g of sucrose.
Place into 250 ml volumetric flask and add 200 ml of UPH2O.
Dissolve and then bring volume to 250 ml.
Filter solution into sterile bottles.
FDU-U Solution
(5-Fluoro-2’-Deoxyuridine (Sigma F0503) + Uridine)
: Used to stop growth and division of glial carpet cells
1. 25 mg FDU
2. 62.5 mg Uridine
3. Pour into beaker
4.
5.
6.
7.
Add 62.5 ml UPH2O  Dissolves Quickly
Syringe filter 0.22 ml into sterile container.
Make 4 ml Aliquots.
Store at –80oC
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Fungizone solution
1. Fungizone (Gibco #15295-017) is dissolved with filtered sterilized H2O.
2. Aliquots to 5ml
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