MDSCs完全培养液中CEE（鸡胚提取物）的自制

tanhehe1006

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：$ Q3 N# ~8 r0 y5 I* \! n+ N
CEE的制备：
1 A! @0 {8 D9 Y* j0 \材料：SPF级种蛋& f) j2 E; a/ N
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。1 @% L7 A! A( s! K, c7 E
2.待到7d左右（大多数选择10-11d），准备提取CEE' _3 q& s# @- P3 x6 T% r# D+ i: W
3.75%酒精清洗卵壳4 f' e8 a) B" \
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）
: R9 @: f7 W' 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
* d6 c# Q" ?% V5 M% i; F% X6.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+ T4 M+ c4 B( m7.除去大的杂质 A：低速离心去除；B：用无菌滤网清除. D. p. M. i& l* M' X4 T# x
8.取上清分装EP管，9000rpm 30min 4℃
/ M0 y8 b9 j' M+ A3 Q7 J9.将所得再取上清12000 rpm 1h 4℃2 K* b% e' L) ^) M- k. w% p1 ]
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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其次来个外文的：
/ n  s/ P9 E4 k( @1. Incubate the chicken eggs for 11 to 14 days at 37°C in a humidified incubator (see Hint #1).
) e2 k6 {2 o) L+ U0 EHint #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. . V! n- B  A( [$ V% {+ |
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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.
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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).
, k$ e3 G1 ~2 e) S3 _  J; f9 u0 ]Hint #2：This should produce approximately 25 ml of volume.
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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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( M3 A) t: u6 o1 R3 X6. Add 100 μl of sterile Hyaluronidase for every 50 ml of embryo/MEM mix. ' {/ \# Z- [9 f/ y' n8 C$ a) z  ^) p- W

! T9 D7 S* W' Q' 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). 9 B" }, |% U3 n+ w/ s6 @4 }

5 ]# b7 ]0 C" k9 M! N; g: r8. 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.

tanhehe1006

0 b7 H4 ~7 B) ^  D- L# J! k

! K7 a: d: u' I  F俗话说有图有真相，哥就来上个过程照片吧。不过是综合的一张。记得做完后要测CEE的活性啊。还有此图是我的制作过程，大家仅供过目啊

qingnuanrenxin

很全啊，真好，以后大家要踊跃上传照片啊

ambassador

以为你在培养基培养出鸡胚了呢！谢谢分享！

maxiaorongqp

很好 很强大

87hgl

请问楼主，CEE一般在什么单位有卖的？有个试剂商说有卖的，进口的10ml要1800，实在是太贵了。

zmf123456

很好很强大！！！！

果子萍

很好

果子萍

想问一下，你的DMEM是高糖的还是低糖的- d  b2 P  J6 B7 V

果子萍

您好，您转入液氮多长时间后再放入37℃水浴中啊？37℃水浴又是多长时间啊？; ]) _' K* p3 k- ?! T2 V1 V