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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：- [. ?5 Y; U3 {" \% F
1 `! C% X& @2 l4 ^6 [首先是我总结的方法：
2 f6 O: k6 \& Z- \3 E1 lCEE的制备：
( t/ ?0 ]: N! l; b4 |/ U* I1 x材料：SPF级种蛋* u8 T4 F- h" S4 \; b% Q( P
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# @; ^# i5 l7 @, J2.待到7d左右（大多数选择10-11d），准备提取CEE
- S+ I" B/ N1 [) M6 N6 y$ Z3.75%酒精清洗卵壳* i* C& k/ B" C0 d' s) w 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）
" l: Z0 }4 Z$ @5 s" T8 J5.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; @) q; K6 [- c* U
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>）% s( P: D0 Z; ] u% j
7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除
1 X2 U) I8 q8 V1 G* B+ s& k6 N d1 f8.取上清分装EP管，9000rpm 30min 4℃# \$ V5 \% I, |, ]! ?
9.将所得再取上清12000 rpm 1h 4℃0 g7 @9 T! A- y8 B1 {) f# T
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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- Z) I; t- n( u- E7 V7 i: b. |3 P其次来个外文的：) Y! Y8 I5 Z+ G& e U8 C/ ]( e
1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1).
( J4 K) s7 {' X, n8 fHint #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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( I3 E' W p' b8 u, T2. Wash the surface of the eggshells carefully with 70% Ethanol.
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1 U# v- ^4 b$ [1 E- X" O3. 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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) ?9 |: a/ j2 _% H u4. 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). # ]! \( Q. h; O5 q* K- y2 k# D; v
Hint #2：This should produce approximately 25 ml of volume.( b8 `7 G0 u" ~- y1 m
6 N* P7 J6 I" G5. 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. 7 w2 w; n% B' I# @
; Z) \8 S4 |; D* W, Q+ [6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix.
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J \$ q/ \1 C% j5 j7. 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. 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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