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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：
. D8 f) W( v* [5 n) b fCEE的制备：
! u, m: p5 q6 Z w, k8 _材料：SPF级种蛋' o8 W( [; e5 q8 L- f7 H2 u% ]: F
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。
( ^! ]: q$ X/ b! m# P. ^2.待到7d左右（大多数选择10-11d），准备提取CEE
1 ~- \1 c' m; K' X- |3.75%酒精清洗卵壳$ A. f6 L* x1 P( V2 E
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 z: q! z" G& ]
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
1 f6 ?+ ?7 B: y8 x5 l, \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>）
2 U: w/ B* Y8 }. M7 }- R2 L7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除
3 U. P/ P2 a9 K( V8 d" G7 I% L8.取上清分装EP管，9000rpm 30min 4℃
% Y |& S/ m/ a0 g4 [. J! n9.将所得再取上清12000 rpm 1h 4℃
# q# v$ \! t6 q" Q* H10.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 K; [5 G3 r! q8 y
4 |# r, `/ y. u0 k: ?4 q" e1 m其次来个外文的：
`( I& k3 S( e# s" e S1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1).
2 o: f' ~" p! ?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. 3 j% o% Z% M7 |7 G
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2. Wash the surface of the eggshells carefully with 70% Ethanol. / A* V2 B' p# g( t, E3 H8 ]
1 n2 H" `. ~" \3 w; x9 @/ y/ o% N% 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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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). $ T; [5 l# d, \7 u1 L7 Q$ D9 l
Hint #2：This should produce approximately 25 ml of volume.! R; g# Q5 g5 ]6 P6 c A4 d
6 O9 _3 A" q; X& E$ ?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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R5 f6 n1 h3 }6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix. + x! d1 W4 F0 h/ M
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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.
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9 m& g6 P0 \0 O5 f# y3 Y9. 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
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