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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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* d! W/ z. P1 K* @, j首先是我总结的方法：
. p7 F `2 K) K. q& uCEE的制备：: T+ K" z- Z/ Y
材料：SPF级种蛋
3 U* l6 _: a6 t$ J7 v% _) z" B4 C8 c1.38℃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。4 R9 _7 D; }; g& k- o' L k2 E
2.待到7d左右（大多数选择10-11d），准备提取CEE% x* p, E/ v7 t T
3.75%酒精清洗卵壳
4 s; \* `6 s3 ~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 T9 _6 }% o/ I4 o( l5.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
Z5 x! @ j1 k% ~. x/ w6.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>）
# ]" r2 O& J) d7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除4 z3 {' d- y! v a6 _9 ]
8.取上清分装EP管，9000rpm 30min 4℃
* T' P) s- | H y2 h7 P9.将所得再取上清12000 rpm 1h 4℃
5 H+ I$ R* ?% X# s: O; {! n10.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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4 @2 }) j1 D0 x其次来个外文的：
+ m1 K7 ^7 W1 t! F; h- h y1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1). 5 H$ M# Z* @# ~- ] u" K6 ~: n
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. 6 V4 r1 E7 } j, Y3 A
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2. Wash the surface of the eggshells carefully with 70% Ethanol. # Q% H/ p4 t; q5 F% ~
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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. ! I( T6 _' D% q& o( J- P5 T4 m
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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). ! M% Z8 ?9 f: ^/ o9 f
Hint #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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5 R( M: v6 |( }( \& y# C6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix. 2 }# k$ y2 Y b) H: ^
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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).
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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.' ]* \- S/ t. j; j
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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 2010-4-21 08:54
很好
