|
  
- 积分
- 193
- 威望
- 193
- 包包
- 618
|
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：
) W. s3 ?9 w6 L1 r) I4 M* h6 Y$ m, A; c% W. F9 w5 o
首先是我总结的方法：* G/ l; I+ p+ h/ N3 [- s
CEE的制备：; U0 ^* B- h! b! l
材料：SPF级种蛋
. c" ^' E% F& ~3 ~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。4 z. _& z* ~+ L8 v' n# _: J$ G) A
2.待到7d左右（大多数选择10-11d），准备提取CEE- ~7 A: B. l$ p" |+ R; T2 c
3.75%酒精清洗卵壳
. c# D4 {* g1 M) d. j- n/ j& u U4.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）2 T# c( |- E: W4 q1 ~
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
2 p! \1 W4 |2 G7 M) C5 B6.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>）
. `/ \# b% E, D! y6 N! J7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除* J! l2 x% A$ U# T8 _
8.取上清分装EP管，9000rpm 30min 4℃8 V/ N" @) B+ g& N
9.将所得再取上清12000 rpm 1h 4℃+ m, Z7 f1 q( E$ m% t0 n
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？）
* E x, m+ n$ `1 h% b3 R1 F0 m9 w" u3 ^, s X
其次来个外文的：
/ U0 L0 A2 d8 ~* c3 p# S! U, t1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1). - v6 E' q. e! @& g. @: V+ P4 R( L
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.
1 l. ]% A8 D( ]5 h; D; e
5 w r t; c0 X8 t9 [2. Wash the surface of the eggshells carefully with 70% Ethanol. + p" O% M5 I4 U# h; p
" ]7 c* G* }1 H1 M0 T3. 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. , _' y" q: P* X9 r) K) A6 x. y
- i( r, i. z5 M8 C: |: b4. 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). ; q7 J: O) a9 i- v- B* {) j
Hint #2：This should produce approximately 25 ml of volume.
6 z. F8 e# }: J- B" a( S5 N! V
1 t1 W& w* B; o4 S0 h5. 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. / \) e1 m, e0 \5 H4 D9 ^1 F9 U
/ O; `( |# o* s1 o+ C1 O) \ }
6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix.
@- A* N4 o' Y1 t" W6 \' U8 B, l w9 }/ j x. I; |+ h" n: U K5 q3 j6 ?. {
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).
9 L3 d% y' `: |0 G) `* `0 t3 e! T" W
8. Filter the supernatant through a 0.45 μm filter and then through a 0.22 μm filter using sterile technique.
1 b8 G; v0 |8 Z* l5 i, {" b; \- a; }: ?% x, A# X2 p; y
9. Aliquot and store at -80°C. |
-
总评分: 威望 + 20
包包 + 20
查看全部评分
|
发表于 2010-4-21 01:10
发表于 2010-4-21 08:54
很好
, |: |% K& c: t' q. f* Z# X$ ]7 z