干细胞之家 - 中国干细胞行业门户第一站

标题: 请问那篇蛋白诱导ips的文章里regular mES cell growth medium的配方是什么？ [打印本页]

作者: mofatuzi 时间: 2012-3-25 09:33 标题: 请问那篇蛋白诱导ips的文章里regular mES cell growth medium的配方是什么？

这篇文章补充材料里提到的) c& {$ u7 F! ?5 L1 \( A4 _4 }& d
http://www.sciencedirect.com/science/article/pii/S1934590909001593
是不是normal mESC growth media再加LIF？

作者: DA_huyang 时间: 2012-3-25 10:31

占个沙发:lol

作者: mofatuzi 时间: 2012-3-25 15:30

我把内容放出来，请各位分析一下，谢谢
Cell culture. }- V6 n' _2 L% R7 g% V
OG2-MEFs were cultured on gelatin-coated dishes in normal MEF media: high-glucose D-MEM
(Invitrogen) with 10% FBS, 0.1 mM non-essential amino acids, and 2 mM L-glutamine. piPS. J# A* b5 `- w1 G+ a# S
cells were cultured on irradiated CF1 MEFs with normal mESC growth media, which consist of7 s3 J: T1 w4 @7 T( q4 A
Knockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 mM 2-ME, 2 mM L-glutamine,
0.1 mM non-essential amino acids, and 1000 units/ml LIF (ESGRO, Chemicon International). The
piPS cells were passaged every 3 days as a single cell suspension using 0.05% trypsin/EDTA* C4 A( p. s" _& K* p1 y) T
and seeded at 1.0x104 cells per cm2 for routine culture. For feeder-free culture, cells are grown
on gelatin-coated tissue culture dishes in chemically defined media, which consist of Knockout
DMEM supplemented with 1xN2, 1xB27, 0.1 mM 2-ME, 2 mM L-glutamine, 0.1 mM nonessential' J+ B1 K/ z( s* C- J+ T
amino acids, 50 μg/ml BSA fraction V (GIBCO), 103 units/ml LIF and 10 ng/ml BMP4, d9 N+ a' F2 C. K3 {! w
(R&D).
Generation of piPS cells; C) }) L% @* H4 }
OG2-MEFs were seeded at 5x104 cells per well in a 6-well plate coated with gelatin in normal
MEF media (DMEM supplemented with 10% FBS). On the next day, media was changed to the% V. A8 ~& X5 |
protein transduction media, which were prepared by mixing the recombinant reprogramming* b( S* C- T8 V4 n& ~
proteins at the final concentration of 8 μg/ml with regular mES cell growth medium/ X3 m. @8 F( i
supplemented with 1000 units/ml LIF. After overnight culture in the protein transduction media,9 t6 V+ g0 H- T5 g1 p1 G
media was changed to normal mESC growth media, and cells were cultured for additional 36
hours before repeating the same protein transduction cycle. Total four protein transduction' s* r- K# v$ p( c# ?
cycles were applied on the cells. After completing protein transduction, cells were then k( k* H. n$ q/ f2 u# y2 a# b( r
passaged onto irradiated CF-1 MEF feeder cells at day 9 in normal mESC growth media. Media
were changed every 3-4 days until GFP+ colonies were observed around day 30-35. GFP+8 `# L/ I4 y+ o- h3 ^. b7 m) X
colonies were then passaged onto new irradiated MEF feeder cells in normal mESC growth
media, and stably maintained and expanded as piPS cells. Some colonies were further selected
and expanded in the presence of pluripotin (1 μM) or PD0325901 (1 μM).

作者: mofatuzi 时间: 2012-3-28 16:50

有知道的吗

作者: song58 时间: 2012-3-30 11:59

normal mESC growth media, which consist of Knockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 mM 2-ME, 2 mM L-glutamine, 0.1 mM non-essential amino acids, and 1000 units/ml LIF (ESGRO, Chemicon International). 4 w0 m# \& F3 Z$ o+ n
人家不是写的挺清楚的嘛

作者: ophden 时间: 2012-3-30 14:07

ES培养液里要加LIF的，我们实验室有人养了很长时间了。

作者: mofatuzi 时间: 2012-4-1 22:29

song58 发表于 2012-3-30 11:59
+ C3 `1 h' C2 e4 |" E4 P3 Znormal mESC growth media, which consist of Knockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 ...

- ^& O* G4 M6 V1 `5 F) v0 K
你复制的是normal mESC growth media。$ d5 {6 V0 @7 A3 W( q- j
后面这个regular mES cell growth medium不知道什么意思，与normal是什么关系？

作者: mofatuzi 时间: 2012-4-1 22:30

ophden 发表于 2012-3-30 14:07 2 H. u, W; A8 ^* m7 A R
ES培养液里要加LIF的，我们实验室有人养了很长时间了。

8 L7 [6 y& Y: h2 Z; w0 I {1 M
我知道要加LIF。只是不知道regular mES cell growth medium和normal mESC growth media是什么关系。

欢迎光临干细胞之家 - 中国干细胞行业门户第一站 (http://www.stemcell8.cn/)