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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：- i0 R6 B& k+ D8 k% A( d
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首先是我总结的方法：0 ?1 _8 N1 W0 n3 Q9 f! V% Y/ }
CEE的制备：
6 H; k" F% f/ C; B材料：SPF级种蛋
! i6 n, r) {0 g/ F; }6 Y1.38℃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。! |8 J$ G( D! z. Z* `. P
2.待到7d左右（大多数选择10-11d），准备提取CEE" ~1 P/ @" e# S
3.75%酒精清洗卵壳" `( v3 _6 p! R
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）4 {+ P( t, G# r4 b
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, B5 i7 v3 f9 y/ [5 [" j
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>）/ i. V1 Q- a% G; \5 b# Y" z
7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除
2 J/ X% S* @; M! Q8.取上清分装EP管，9000rpm 30min 4℃
* s/ z& S2 g9 U( u9.将所得再取上清12000 rpm 1h 4℃* ]5 e# q9 `7 ]% l6 |9 `1 t8 [
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？）2 Z! I7 [9 W% H X4 q5 v
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其次来个外文的：
8 P& n2 z$ B# @1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1). . I- {/ E; J$ q' `% R% e
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.
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, n4 Z1 ~, j, V2. Wash the surface of the eggshells carefully with 70% Ethanol.
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" z' @, k8 j$ y5 j5 u3. 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. 2 t2 z/ }6 p) l2 U, U2 a
" q! o: t# o" |& ^4 C4. 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 n. g# a1 B0 p* |# c* XHint #2：This should produce approximately 25 ml of volume.
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5. 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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! _$ U1 _% d5 T/ p6 n0 V6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix.
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% ? U/ B9 z& z7. 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). ! m0 U& f% n& J1 _6 @) j' @7 U8 A" b
. v$ G; V& D5 p0 j5 L9 V. B% {8. Filter the supernatant through a 0.45 μm filter and then through a 0.22 μm filter using sterile technique.. k( @6 c' {" P! o/ \4 k
) @8 u, Z: r0 ]" K- H" i: y$ B( P9. 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
