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看过众多有关MDSCs分离的朋友应该度看到其中培养体系中的CEE。下面就介绍一下自制的方法：6 g, S5 z7 T3 W: _4 W7 b
) x$ {& _% o) r3 B9 W首先是我总结的方法：! r: I5 A7 {/ Z' [2 b& T: [7 h
CEE的制备：
7 U/ p8 s% N) Q: H7 b" P- b材料：SPF级种蛋
) e, F$ J6 u! z2 I5 d1.38℃烘箱孵育，保持一定湿度环境，勤翻蛋，时时用手电观察鸡胚发育情况。
# W$ e6 @8 I0 ]9 Q4 [2.待到7d左右（大多数选择10-11d），准备提取CEE; P9 H1 G3 x& C- I& L, H
3.75%酒精清洗卵壳4 E( ?# e& [" G/ v
4.由气室出发，小心剪开蛋壳，剥除膜，解剖出鸡胚置入无菌平皿，去除鸡眼后经PBS充分清洗血液，卵黄，然后置入4℃DMEM（3个/7ml）（先充分冲洗，需要剪碎）! i! t Q/ @' n8 V p$ ^: w! }
5.充分泡软后，混匀（A：waring blender搅拌器<文献述>；B：用大号注射器混匀<需要让组织十分碎>）约可得到25ml/10个EE2 L. C' V8 X' }* N. S# E1 k& f
6.加入等体积4℃DMEM，继续混匀（A：继续用注射器，可由大到小的顺序<不靠谱>；B：先转入液氮冷冻，后37℃水浴，共2次<当细胞70%左右为单细胞后方可进行此步骤>）
) x! A2 C& ?4 d4 n7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除
6 p% y: N2 k! c: }) i8.取上清分装EP管，9000rpm 30min 4℃ \9 v; Y8 k; f7 b
9.将所得再取上清12000 rpm 1h 4℃
; ~1 l5 b6 h- b& H9 a# y10.取上清，经0.45or/&0.22um过滤（上述操作尽量无菌，CEE相当容易堵膜），-80℃分装保存（是否加两性霉素？）7 V; W+ P1 p M: [
3 M: @% Z. t2 ?/ Y其次来个外文的：
4 M! d: l% ]( A) F9 \1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1).
) W2 W# N! ]" M* c/ O+ v# l1 W4 nHint #1：The length of time that the eggs are incubated depends on the age of the eggs when they arrive at the lab. Normally, eggs that are ordered arrive within 2 to 3 days of being laid. Most universities and educational institutions have agreements with neighboring or local farms. Please check with your animal facility at your institution for a source for fertilized chicken eggs.
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2. Wash the surface of the eggshells carefully with 70% Ethanol.
# `1 y7 C' S# |: {$ v6 ]- M G! X) n ]% a
3. Crack open the egg. Dissect out the embryos and place them in MEM at 4°C. Use approximately 7 ml of MEM for every 3 dissected embryos. * j- ]: d9 C- I' U
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4. Macerate approximately 10 embryos at a time by passing them through a 30 ml syringe into a 50 ml sterile centrifuge tube (see Hint #2). 6 Z- [" b) |! U: T- `, ?& `
Hint #2：This should produce approximately 25 ml of volume.8 P9 }* P: R8 }" Y) N- a; G/ S" Y
% E5 ^/ m0 ?, t+ X1 q5. Add an equal volume of MEM at 4°C to the tube of embryos. Incubate with rotary shaking action for 45 min at 4°C.
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# d$ v4 C: `8 N. n# h6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix.
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7. Centrifuge the mix at 30,000 X g for 6 hours at 4°C (16,000 rpm for 6 hours using a Sorvall™ SS34 rotor). ! x" k8 t/ B$ N( B n
- D' o. W/ r' U) {8. Filter the supernatant through a 0.45 μm filter and then through a 0.22 μm filter using sterile technique.
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( l5 q3 S, P6 m% q. \0 |9. Aliquot and store at -80°C. |
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发表于 2010-4-21 01:10
发表于 2010-4-21 08:54
很好
