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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：" h8 F8 ]: h. h9 [1 [& i2 b3 l7 K
6 `0 a$ ~! c+ F6 \9 y0 m首先是我总结的方法：2 R, O0 c0 p f4 F1 s
CEE的制备：
3 R; p2 m: ]# P, u8 \材料：SPF级种蛋
, q: Z7 ]1 B0 o0 X1.38℃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 O; d3 R" C2 z6 X. k2.待到7d左右（大多数选择10-11d），准备提取CEE
% x6 ~6 S4 ]& q- _3.75%酒精清洗卵壳; N& q0 c8 k* I* X" N
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）$ D* J( `8 W1 T1 c# S
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! O; l! y! g9 S# d* K
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>）, g/ ~2 O6 k; \% E+ X2 ]& b) n
7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除: H" K" G5 L3 O, H3 w
8.取上清分装EP管，9000rpm 30min 4℃
9 g* t/ J7 F9 [& Z0 Q$ J: z2 y! s5 A9.将所得再取上清12000 rpm 1h 4℃
# u4 C, N4 d9 U8 h* j [. 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？）: O. o4 t0 H& K- V
! f$ G+ {7 Z* }其次来个外文的：
9 j, R9 D. k4 B6 E5 Q- x1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1).
' j2 _/ M) @3 \5 L$ hHint #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. . }9 \( D8 X' h, X8 K
1 H- d8 o/ L1 k: K! |3 X2. Wash the surface of the eggshells carefully with 70% Ethanol.
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0 x: F! T J0 ~; X( a j3. 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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+ u& J5 S- [1 p6 J) y( ~. W5 S4. 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). 6 q6 `* A2 H# V. o
Hint #2：This should produce approximately 25 ml of volume.5 ~7 s8 r8 U6 ]8 ~( e. T
- ?( u1 V2 q$ s: l$ x3 S5. 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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6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix. / K$ ]( ~8 K$ w1 u! r! [" J! f
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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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: c, o7 b$ c; g' E8. Filter the supernatant through a 0.45 μm filter and then through a 0.22 μm filter using sterile technique.% }: J% E7 w& J1 z$ d3 C- N/ N) ]
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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
