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我把内容放出来，请各位分析一下，谢谢
+ x' ~( k! A* S0 C2 n, tCell culture! g% U, }+ V- t
OG2-MEFs were cultured on gelatin-coated dishes in normal MEF media: high-glucose D-MEM
4 U, D0 O6 F' R; l4 Q: }# l(Invitrogen) with 10% FBS, 0.1 mM non-essential amino acids, and 2 mM L-glutamine. piPS' q1 F8 N1 Z$ a- p& @9 g V
cells were cultured on irradiated CF1 MEFs with normal mESC growth media, which consist of3 ?& n P3 B* |
Knockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 mM 2-ME, 2 mM L-glutamine,# c9 Q' j5 F( A) N) |
0.1 mM non-essential amino acids, and 1000 units/ml LIF (ESGRO, Chemicon International). The8 p6 m( M8 N; V
piPS cells were passaged every 3 days as a single cell suspension using 0.05% trypsin/EDTA
D1 m; T( s: ? J2 {8 Q$ H) fand seeded at 1.0x104 cells per cm2 for routine culture. For feeder-free culture, cells are grown* u" k7 [. O7 e$ D5 p& `6 h
on gelatin-coated tissue culture dishes in chemically defined media, which consist of Knockout T4 H2 D+ V' w5 X/ D
DMEM supplemented with 1xN2, 1xB27, 0.1 mM 2-ME, 2 mM L-glutamine, 0.1 mM nonessential7 f9 e" V7 \9 _
amino acids, 50 μg/ml BSA fraction V (GIBCO), 103 units/ml LIF and 10 ng/ml BMP4. r$ g0 P- t, c' z6 q+ n" S
(R&D).
' N' d2 \6 f: U# k, ?& C }Generation of piPS cells0 {5 s$ c# D* e/ g
OG2-MEFs were seeded at 5x104 cells per well in a 6-well plate coated with gelatin in normal$ F8 W! q" {1 l% F0 F5 E. l' r) b
MEF media (DMEM supplemented with 10% FBS). On the next day, media was changed to the" U: C8 C# K4 y& @
protein transduction media, which were prepared by mixing the recombinant reprogramming
2 Y0 H3 d' M+ l0 `) C6 i3 {9 }: |proteins at the final concentration of 8 μg/ml with regular mES cell growth medium
; F1 |$ [* v. S+ F4 I! }# ysupplemented with 1000 units/ml LIF. After overnight culture in the protein transduction media,
) q4 W9 Z! g5 Ymedia was changed to normal mESC growth media, and cells were cultured for additional 36 [% R, m. Y G. @1 |$ X2 X
hours before repeating the same protein transduction cycle. Total four protein transduction% o. n# T5 A: W
cycles were applied on the cells. After completing protein transduction, cells were then9 ~. R/ h \7 C* X, @
passaged onto irradiated CF-1 MEF feeder cells at day 9 in normal mESC growth media. Media+ R7 \. c3 h1 q t
were changed every 3-4 days until GFP+ colonies were observed around day 30-35. GFP+
1 F' E+ q n8 w2 r9 N% D& vcolonies were then passaged onto new irradiated MEF feeder cells in normal mESC growth
' T h& q& j" J5 c$ V1 @& z$ g6 X& Xmedia, and stably maintained and expanded as piPS cells. Some colonies were further selected _2 Q) `2 v7 X. H3 Y
and expanded in the presence of pluripotin (1 μM) or PD0325901 (1 μM). |
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发表于 2012-3-25 09:33
