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标题: 小鼠胚胎成纤维细胞的制备及处理(饲养层) [打印本页]

作者: qgjin 时间: 2010-5-7 04:20 标题: 小鼠胚胎成纤维细胞的制备及处理(饲养层)

本帖最后由 qgjin 于 2010-5-7 04:30 编辑
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PREPARATION OF MEF CULTURES0 e& y$ C5 W; X, Q
1. Remove embryos (E13–E16) from a pregnant mouse, rinse in PBS, remove and discard
placenta and fetal membranes, head, liver and heart. Retain and rinse the carcasses in PBS./ m! U3 n1 O$ J, k0 x$ i( y
2. Mince the embryonic tissue in 5 ml of trypsin solution, transfer to an Erlenmeyer flask containing a stir bar and a few 5mm glass beads, and stir on& i4 Y3 Q+ v3 M
magnetic stirrer for 15–25 min (use shorter incubation times if the embryos are E13 and use longer incubation times if the embryos are E16). Ideally,$ t$ q1 g4 F1 l! ?/ m
the resulting cell suspension should be essentially free of any larger pieces of tissue and should not be too viscous (genomic DNA-lysed cells).5 X( N) ~8 `* M* F& |$ X
3. Pipette the cells with a 2-ml glass pipette to achieve cell suspension. Filter the suspension through a sieve or a screen, add 10 ml of
MEF growth medium and centrifuge at 450g for 5 min at RT. Resuspend the pellet in about 3 ml of MEF growth medium.* I4 L5 W z- `' x
MEF growth medium: MEF growth medium DMEM (4.5 g/ liter glucose) supplemented with 15%
(vol/vol) FBS and 1%penicillin/streptomycin solution for cultivation of MEFs.Store at 4 ℃ for up to 2 weeks.8 J- h/ Z/ J- L$ s% d+ o# E
4. Plate the cell suspension onto cell culture plates at a density of about 2*106 cells per 100 mm plate (P0; i.e., passage 0) and incubate in
MEF growth medium at 5% CO2 and 37℃ for 24 h.
5. After 24 h, change the medium to remove debris, erythrocytes and unattached cellular aggregates.
6. Cultivate for an additional 1–2 d until the cells reach B90% confluence.
7. Rinse the MEF plate with Ca2+- and Mg2+-free PBS twice.
8. Add trypsin solution to the culture plates and incubate for 1–2 min.1 k& Z6 t) ?7 i6 f+ E
9. Aspirate trypsin solution, collect cells in MEF growth medium and expand them once (1:3 split).
10. Freeze any MEFs not needed immediately.
-POINT MEFs at P0 can be stored in liquid nitrogen up to 1 year.
11. Continue to passage cells as described in Steps 6–9 of this Box. MEFs at passages 2–4 are most suitable as feeder layer for cultivation of, a4 }+ Z1 e; ]0 d
undifferentiated maGSCs; prepare as follows.
12. Incubate a confluent plate of MEFs with medium containing mitomycin C (10 ug/ ml) at 37 ℃ for 3 h.7 b2 x2 ~( n4 [/ g0 o5 A7 O
13. Aspirate the mitomycin C solution and wash three times with PBS.
14. Trypsinize MEFs as described in Steps 7–9 of this Box and replate them on new gelatin (0.1%)-treated microwell plates or to Petri dishes
at a density of 50,000–60,000 cells/ cm2.
-CRITICAL STEP MEFs prepared 1 d before maGSC subculture are optimal. Although established cell lines may grow well on 2- to
3-d-old MEFs, it is critical to use MEFs within 1 d after mitomycin C treatment for primary cultures (early derivation steps, mechanical
passaging and thawing).

作者: ppcl2 时间: 2010-5-7 09:50

俺感觉这个protocol不咋地哈，有几个人用来自E16胚的MEF来做feeder？) E; A S0 a, `: R8 S% v. d
remove and discard placenta and fetal membranes, head, liver and heart.这个也粗放了点哈！
E16那么多细胞，3ml培养基，不是开玩笑吗？

作者: w1986725 时间: 2010-5-7 10:25

都没有人用胶原酶处理的吗？我想知道胶原酶处理的时间啊！~

作者: ppcl2 时间: 2010-5-7 12:31

回复 3# w1986725 & k6 ?, W1 R n( ?2 r2 f( l/ w0 X
差不多，也是15到20分钟左右。

作者: w1986725 时间: 2010-5-7 13:07

请教一下，是用胶原酶Ⅱ还是Ⅳ？

作者: qgjin 时间: 2010-5-7 16:05

本帖最后由 qgjin 于 2010-5-7 16:21 编辑 9 b0 ^0 F% x8 f' t' k9 H; j

俺感觉这个protocol不咋地哈，有几个人用来自E16胚的MEF来做feeder？
/ B+ W' ^* i5 `' Mremove and discard placenta and fe ...
1 `! ^& ?. r( o$ c( p7 Y! Hppcl2 发表于 2010-5-7 09:50

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只是在论文中搜到的，请讲细一些好吗？（譬如：E？胚最合适？；取哪个部位的细胞效果更好，怎么取？；终止消化要添加多少培养液？为什么？）我是新手不太懂请教一下。

作者: ppcl2 时间: 2010-5-8 01:07

回复 6# qgjin
Feeder一般用E12.5或E13.5，最迟不超过E14.5，俺取的MEF都是去头，掐尾，去四肢，内脏全去掉，剩下的躯干剪碎，再0.25%的含EDTA胰酶消化20分左右，加含FBS培养基吹打均匀并停止反应，离心，用吸管小心去除大多上情，再用10多毫升培养基重悬，培养，第二天换液，然后一直养到差不多满了。

作者: majing217 时间: 2011-7-6 16:17

顶楼上，我基本上用13.5天的做饲养层

作者: llecstem 时间: 2011-7-26 14:30

回复 ppcl2 的帖子0 i C7 b$ B9 k( G1 P

想问一下，为什么去掉头内脏四肢？如果说头和内脏的细胞类型太多，所以要去掉，那四肢呢？谢谢！

作者: hcluo 时间: 2011-7-26 19:21

我个人觉得有一个时间范围，基本都可以使用，但不能太超过。

作者: majing217 时间: 2011-7-28 09:26

我通常取13.5天的，也是去头去尾去内脏去四肢，也是用胰酶消化15分钟左右，然后用含FBS的培养液终止，一般酶与培养液的比例为1:2.之后一只胎鼠接种到一个大皿中。

作者: 褚cell 时间: 2011-8-4 23:11

不知道大哥验证过没啊！建议谁能把做的MEF细胞照片展示一下，这样大家交流一下怎么才能做得好看并纯化MEF细胞。

作者: dongxin 时间: 2011-9-1 15:30

本帖最后由 dongxin 于 2011-9-1 15:31 编辑
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取12.5d—13.5d的小鼠胚胎，将孕鼠断颈处死；取出子宫，将含有胚胎的子宫置于干净的100-mm培养皿中，将子宫用PBS清洗三次，去除血迹；分离胚胎，去掉小鼠的头部、肝部、四肢；剪碎，0.05%胰酶消化；用 MEF培液终止并吹打组织块；用70um滤器过滤；离心，一般1个胚可培养一个35-mm培养皿。供参考，祝好运！8 V( D+ W5 Q& E

作者: irise1006 时间: 2012-3-9 17:10

ppcl2 发表于 2010-5-8 01:07 / v, I1 @5 o6 D n; r% P$ F2 c
回复 6# qgjin
5 D1 t) y) ~9 K2 `1 b/ C3 UFeeder一般用E12.5或E13.5，最迟不超过E14.5，俺取的MEF都是去头，掐尾，去四肢，内脏全去 ...

是这样，但胰酶消化时间要根据你买的厂家，这个掌握好，别消化过！

作者: irise1006 时间: 2012-3-9 17:10

13.5较好！

作者: qwch1988 时间: 2012-3-29 22:35

回复 w1986725 的帖子# g( Z" y$ J, e% |& h9 B* I
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好像一般都用胶原酶Ⅳ

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