Primary Hippocampal Neuron Culture(프라이머리 히포캠퍼스 뉴런 컬처) - 1

Primary hippocampal neuron culture에 대한 정리입니다. 뇌질환 연구를 위한 마우스 실험입니다. 대략적으로 생각나는대로 스윽스윽 정리했습니다. 많은 도움이 되길바라겠습니다.

 

먼저, material들과 equipment 입니다.

 

재료들

Boric acid

Borax (Sodium tetraborate decahydrate)

Poly-D-lysine hydrobromide

Mininal Essential Medium (MEM) variation : Liquid

D-(+)-Glucose

10x HBSS, calcium, magnesium, no phenol red

HEPES 1M, pH 7.33

Sodium Pyruvate (100 mM)

Penicillin-Streptomycin (10,000 U/mL)

Phenol-red

Bovine Serum Albumin (BSA)

DNAse

Fetal Bovine Serum, qualified, Canada (FBS)

200mM, L-glutamine (GlutaMAX Supplement)

B-27 Supplement (50X), serum free

Neurovasal Medium, no phenol red

 

장비

Autoclave

Ultrapure water purification system

Clean bench

수술도구

Incubator

Shaking machine

Refrigerator

Confocal Microscope

Centrifuge

 

0.1M Borate Buffer, pH 8.5,Store at 4℃	사용된 양
Boric acid	0.775 g
Borax (Sodium tetraborate decahydrate)	1.225 g
Water	225 mL
Mix, Check pH 8.5, Fill up to 250mL, 0.22μm Filtering

 

10mg/mL PDL stock, 125μL aliqout, Store at -80℃	사용된 양
Poly-D-lysine hydrobromide	0.05 g
Water	5 mL

Working용은 50μg/mL borate buffer에 희석 사용 (1/200 -> borate 24.875mL + 10mg/mL stock 125μL = 25mL)

 

20% glucose in MEM (20g/100mL), 15mL aliqout, Store at -80℃	사용된 양
Mininal Essential Medium (MEM) variation : Liquid	35+a mL
D-(+)-Glucose	10 g

Glucose 넣고 바로 흔들어서 녹여야 함
 MEM으로 Final volume맞춤 (Conical tube 50mL line에 맞춤)

 

HBSS w/ phenol-red, Store at 4℃ for up to 2 months	사용된 양 (mL)
10x HBSS, calcium, magnesium, no phenol red	50
20% glucose in MEM (Final 2.5%)	12.5
HEPES 1M, pH 7.33	5
Sodium Pyruvate (100 mM)	5
Penicillin-Streptomycin (10,000 U/mL)	5
Water	422.5
Phenol-red(나중에)	0.0055 g

10N NaOH 10 μL 넣고 pH 7.33 가깝게 조정함.
0.22 μm Vacuum filtering.

 

BSA, aliqout, store at 4℃ for up to 2 months	10mL	50mL
HBSS w/phenol-red	8 ml	40 ml
Bovine Serum Albumin (BSA)	1 g	5 g

BSA powder는 부피가 좀 커서 2ml HBSS에 1g을 넣어주고 rocker로 녹이고 HBSS 추가. 거품이 생겨 주의해야함. 다 녹으면 1N NaOH 350 ul정도 넣어서  pH 7.33

 

DNAse, aliqout, store at -20℃	1 mL	5 mL
DNAse	0.005 g	0.025 g
Water	1 ml	5 ml

 

Trituration soluton, aliqout, store at 4℃ for up to 2 weeks	20 mL	50 mL
HBSS w/ Phenol-red	15.8 ml	39.5 ml
Fetal Bovine Serum, qualified, Canada (FBS)	2 ml	5 ml
10% BSA	2 ml	5 ml
200mM, L-glutamine (GlutaMAX™ Supplement)	100 ul	250 ul
0.5% DNAse	80 ul	200 ul
pH 7.33 조정. 1N NaOH 1uL를 넣고 리트머스로 pH확인. BSA solution 쓸 경우 3 ul, 0.22uM filter

 

Plating media aliqout, store at 4℃ for up to 1 week	50 mL	500 mL
Starting Medium	85 ml	42.5 ml
Fetal Bovine Serum, qualified, Canada (FBS)	12 ml	6 ml
B-27™ Supplement (50X), serum free	2.4 ml	1.2 ml
200mM, L-glutamine (GlutaMAX™ Supplement)	600 ul	300 ul

 

Neurobasal media aliqout, store at 4℃ for up to 1 week	100 mL	20 mL
Neurobasal™ Medium, no phenol red	97.75 ml	19.55 ml
B-27™ Supplement (50X), serum free	2 ml	0.4 ml
200mM, L-glutamine (GlutaMAX™ Supplement)	250 ul	50 ul

 

일단 material들을 나열하다 보니 생각보다 많네요

 

실험은 상황에 따라 다를 수 있으니 참고만해주세요~

 

필요한 정보가 있으시다면 물어보셔두됩니다!

 

Protocol 다음 포스팅에 이어서 작성하겠습니다.