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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： \$ i- b8 ?; z. L% ^, C
0 x" z- D1 b. n. J0 h- X* H0 @首先是我总结的方法：
a6 n1 ] S4 L1 H7 k# N9 e0 r1 [( DCEE的制备：
0 u. ?. k: K; c+ ]材料：SPF级种蛋+ P# b' Z/ R: |- c. h
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。
! L, N( L7 M9 }1 L3 E2.待到7d左右（大多数选择10-11d），准备提取CEE
* w4 n9 v3 u P4 b) u3.75%酒精清洗卵壳( V- F# d% V8 X# l* D
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）
, V5 Z4 ~8 r3 i# y+ G1 u$ M5.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* t! o5 [* k) x
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>）" c( M& u+ Z1 U
7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除
: [& ~( M) c" }6 r E9 ~8.取上清分装EP管，9000rpm 30min 4℃
! K0 S9 C. K; T" c% V m& k" {9.将所得再取上清12000 rpm 1h 4℃6 F8 _! h3 D. R' q- v3 ]
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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F. a |5 [ r8 E I其次来个外文的：% n# ^8 Q! e& S% H, |4 S
1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1).
( o5 E0 N1 l* X5 R( c8 JHint #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. ( b6 G8 s# u( \
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2. Wash the surface of the eggshells carefully with 70% Ethanol.
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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. / O& x8 k1 ?3 k5 A( e
v8 x' K4 ~( F2 L3 T4. 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). . A5 k4 H: D1 ]: z
Hint #2：This should produce approximately 25 ml of volume.# ^" C) ~3 D; o4 Q$ c% ^9 o+ _, E# b
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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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' R$ G6 m0 p/ j1 \0 w: ~% r6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix. 6 ?$ c: J( U, B. f& u1 b+ b
3 z6 |" _; m5 k7. 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). 5 ~- T- R. h; E+ m
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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.0 d* Y& `- S6 ^; A0 m1 x; W3 O" ]
& e: {$ A5 V: v# g1 \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
