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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：
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首先是我总结的方法：
7 C; ]% l* G. M- N$ \. e' l/ f c6 Y, hCEE的制备：
8 A4 r7 a+ }; b* A5 M材料：SPF级种蛋# s8 \" ^, ^$ a. N8 v" k& b7 I6 m
1.38℃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。
: y( U s6 o0 `0 K! p2.待到7d左右（大多数选择10-11d），准备提取CEE* w7 H- c1 ^1 `( R1 t
3.75%酒精清洗卵壳
& N {4 N% b7 ^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）! B, S9 J4 h9 {! R, b0 n3 W& I8 ]
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EE$ ]' [7 ?- ]6 O1 K/ j1 R
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>）
3 s" z" n _0 L# G7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除, s# s$ U# N( p; {: u0 F
8.取上清分装EP管，9000rpm 30min 4℃5 k$ G- h& ~- t
9.将所得再取上清12000 rpm 1h 4℃$ P6 a" g0 R, T# N9 P) o9 E
10.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？）
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其次来个外文的：0 E2 `% k7 I; \. S' M
1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1). + W# D9 T8 c# P$ B
Hint #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. ) J8 I2 m7 z5 R
4 q/ E& \9 f+ I2 p2. Wash the surface of the eggshells carefully with 70% Ethanol. Q/ O* x- _5 ?
2 i$ ? ?0 y1 }( d3 n0 C/ s# i3. 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.
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5 Z5 G Y8 D9 C* [4 E/ }$ z4. 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). 1 o& d# A, [. F B
Hint #2：This should produce approximately 25 ml of volume.
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$ v% K- U& h( E5 m' Y+ K5. 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. & h( d( M4 U8 ~0 x" A7 S
$ O8 P$ X4 |* f6 U8 M6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix.
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# F t5 y/ W1 A9 S# o) {- m7. 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). 2 s' h& |" v0 G' x4 d K
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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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9. Aliquot and store at -80°C. |
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发表于 2010-4-21 01:10
发表于 2013-9-26 11:16
发表于 2013-9-26 10:55
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很好
