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标题:[求助]大鼠微血管内皮细胞培养

tuuu2[使用道具]
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发表于 2012-4-9 15:03 资料 个人空间 短消息  加为好友 

 小中大

[求助]大鼠微血管内皮细胞培养




最近在养大鼠脑微血管内皮细胞，弄的焦头烂额的，在文献里看到的那些方法，试过了都不行。 不知有没有在北京的高手正在养这种细胞，可不可以去贵实验室学习一下？或者能有偿赠与我一瓶P1或是P2代的细胞呢？
个人信息：边雨竹，清华大学微生物实验室。
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sunnyB[使用道具]
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发表于 2012-4-9 15:18 资料 个人空间 短消息  加为好友 

 小中大

我也正在养大鼠的BMEC，P0和P1代都没问题，P2代就死了。。。
大概是因为没加ECGS
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zsxan1990[使用道具]
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发表于 2012-4-9 15:18 资料 个人空间 短消息  加为好友 

 小中大

我养的是心脏微血管内皮细胞，不知道在培养方法上是不是相似？我养的细胞倒是还可以
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vvmmoy[使用道具]
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发表于 2012-4-9 15:21 资料 个人空间 短消息  加为好友 

 小中大
我也正在养大鼠的BMEC，P0和P1代都没问题，P2代就死了。。。
大概是因为没加ECGS

————————————————————————————————————————————————————————————————

我现在还取不出来，不知这位同学用什么方法得到P0代细胞的，能不能介绍一下？用的多少天的大鼠，怎样的消化和分离方法等等。谢谢
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vivian4123[使用道具]
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发表于 2012-4-9 15:21 资料 个人空间 短消息  加为好友 

 小中大

Reagents & Consumables
1.  DMEM (Invitrogen, Cat#11965-092)
2.  Fetal bovine serum (Hyclone, Cat #CH30160.03)
3.  Glutamine (GIBCO, Cat #21051-024)
4.  Penicillin10.000U/ml streptomycin 10.000μg/ml (GIBCO, Cat #B-13234)
5.  Collagenase IV (Invitrogen, Cat# 17104-019)
6.  Trypsin (Invitrogen, Cat # 27250018)
7.  EDTA (Sigma, Cat# E6758)
8.  HBSS (Ca, Mg-free) ( (Sigma, Cat#H4891)
9.  HBSS (Sigma, Cat#H1487)
10.  Sodium Pyruvate (Invitrogen, Cat#11360070)
11.  Heparin
12.  Non-essential amino acids (Sigma, Cat#M7145)
13.  100mm dish (Corning, Cat# 430166)
14.  Collagen type I (BD, Cat# 354236)
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vivian4123[使用道具]
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发表于 2012-4-9 15:22 资料 个人空间 短消息  加为好友 

 小中大

1.  Culture medium
DMEM: 90%,
FBS: 10%.
Supplements: L-glutamine 2.0 mM, Penicillin100U/ml, streptomycin 100μg/ml, 1% non-essential amino acids, sodium pyruvate, Heparin 5U/ml. Store at 4oC.

2.  0.1% collagenase IV: Dissolve in HBSS, store at 4oC

3.  25% BSA: Dissolve in HBSS (Ca, Mg-free), sterilize the solution by passing through a 0.22 µM membrane filter; store at 4 oC.

4.  Isolation medium:
DMEM: 98%,
FBS: 2%.
Supplement: 5%Penicillin-Streptomycin

5.  Ethanol/Collagen solution: Dilute collagen I to 0.9mg/ml with filtered 60% ethanol and mix thoroughly. Store at 4 oC and use within 1 week.
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vivian4123[使用道具]
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发表于 2012-4-9 15:22 资料 个人空间 短消息  加为好友 

 小中大
Methods
Isolation of Rat Brain Microvessel Endothelial Cell
Prepare collagen-coated plates.
1.  Dilute the collagen type I to 0.9mg/ml with filtered 60% ethanol.
2.  Add 5ml per 10 cm plates. Allow the plates to dry uncovered overnight in a laminar flow hood under UV light. The coated plates can be stored in the hood for 1 or 2 days.
3.  Wash with PBS buffer 3 times before plating the cells.

Isolation Procedure
1.  Take 2 rat cerebral cortices.
2.  Wash cortices in HBSS.
3.  Dissect free of meninges and white matter in HBSS.
4.  Put the tissues to isolation medium.
5.  Mince tissue with surgical blades until it uniform 2-3mm bits.
6.  Transport the organization levitation medium into homogenizer, homogenized the medium till it looks like milk shake.
7.  Transfer the medium into 50 ml tube and centrifuge (5 min, 250g, 20 oC).
8.  Resuspend the pellet in 20 ml 25%(w/v) BSA.
9.  BSA gradient (1000g, 15 min, 4 oC).
10.  Discard myelin floating on BSA and resuspend the pellet containing the microvessels in 5 ml of 0.1% collagenase.
11.  Digest for 30 min at 37 oC, shake gently every 15 min.
12.  Stop digestion by adding isolation medium.
13.  Pellet the tissue (250g, 5 min).
14.  Resuspend the pellet in 20 ml 25%(w/v) BSA.
15.  BSA gradient (1000g, 15 min, 4 oC).
16.  Resuspend the pellet in culture medium and plate out.
17.  Culture at 37 oC, 5% CO2 until confluence.
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vivian4123[使用道具]
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发表于 2012-4-9 15:23 资料 个人空间 短消息  加为好友 

 小中大
Isolation of Astrocytes
Prepare poly-D-lusine-coated plates.
1.  Dilute the poly-D-lysine solution to 10μg/ml with sterile Milli-Q water.
2.  Add 5ml per 10 cm plates. Allow the plates to dry uncovered overnight in a laminar flow hood under UV light.
3.  Wash with PBS buffer 3 times before plating the cells.

Isolation Procedure
1.  Use 6 rat pups (2 pups/flask), of one/two days old.
2.  Transport them in a clean Petri dish instead of in their cage.
3.  Dip the rats in 70% alcohol and lay them in a Petri dish.
4.  Decapitate the rats.
5.  Remove the brains and transport them to a sterile Petri dish, containing of cold DMEM buffer.
6.  Isolate the cortex with sterile forceps and remove the meninges. Cut thecortex in pieces and put them in a 50ml tube with 10ml DMEM buffer, 37 oC.
7.  Add 5ml trypsin-EDTA, 37oC to the 10ml suspension resulting in a final concentration of 0.1% trypsin.
8.  Incubate in a shaking waterbath (80 rpm, 37 oC, 25 min).
9.  Add DMEM +10% FBS to stop trypsinization to a total volume of 50 ml.
10.  Spin the cell suspension for 5-8 min at 25g.
11.  Resuspend the pellet in 10 ml DMEM + 10% FBS.
12.  Filter the cell suspension through a 45 μm mesh and rinse with 10 ml DMEM +10% FBS.
13.  Seed the cells in 10 cm plate.
14.  Culture at 37 oC, 5% CO2 until confluence.
15.  When confluent (after 8 days) shake the culture flasks in a shaking waterbath at the highest speed (250rpm) overnight, then tefresh the medium. In this way the type 1 astrocytes are separated from type 2, oligos and microglia, because astrocytes are firmly adhered to the culture dish while the oligos and microglia are not.
16.  Culture at 37 oC, 5% CO2 until confluence, then wash the cells with PBS.
17.  Passage with 5 ml trypsin-EDTA, shake and tap until most of the cells detach.
18.  Pool and add culture medium to stop trypsinization.
19.  Spin the cell suspension for 5 min at 125g.
20.  Resuspend the pellet in culture medium and plate the cells in poly-D-lysine-coated dishes.
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vivian4123[使用道具]
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发表于 2012-4-9 15:23 资料 个人空间 短消息  加为好友 

 小中大
出处：Drug transport across the blood-brain barrier: in vitro and in vivo techniques 作者：Albertus G. De Boer, Win Sutanto
google book 上有preview,稍作改动以迎合我们现有条件。
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vivian4123[使用道具]
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发表于 2012-4-9 15:25 资料 个人空间 短消息  加为好友 

 小中大

大家的原代微血管内皮细胞养得怎么样了??
我也要养,先来报个到!
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