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我把内容放出来，请各位分析一下，谢谢% W( f G4 o- N' G3 n
Cell culture9 v9 e/ f8 `* o
OG2-MEFs were cultured on gelatin-coated dishes in normal MEF media: high-glucose D-MEM
, ^$ R/ W. Q" p- \3 N3 N5 t(Invitrogen) with 10% FBS, 0.1 mM non-essential amino acids, and 2 mM L-glutamine. piPS$ x4 {* k* A6 t. J
cells were cultured on irradiated CF1 MEFs with normal mESC growth media, which consist of g! b; m- x) ~. f Z4 }
Knockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 mM 2-ME, 2 mM L-glutamine,
' ]* A8 B7 h7 L7 ~* m! `6 D3 A0.1 mM non-essential amino acids, and 1000 units/ml LIF (ESGRO, Chemicon International). The
7 `( v6 D7 A0 }2 F8 I' JpiPS cells were passaged every 3 days as a single cell suspension using 0.05% trypsin/EDTA5 O3 m8 d8 U' i) s2 b$ L
and seeded at 1.0x104 cells per cm2 for routine culture. For feeder-free culture, cells are grown
! Q$ V [: Y4 |" X) i& Con gelatin-coated tissue culture dishes in chemically defined media, which consist of Knockout {0 [) U) X7 j! P. v1 i
DMEM supplemented with 1xN2, 1xB27, 0.1 mM 2-ME, 2 mM L-glutamine, 0.1 mM nonessential9 x! c' l. @6 m/ p% S
amino acids, 50 μg/ml BSA fraction V (GIBCO), 103 units/ml LIF and 10 ng/ml BMP4
$ f- l# I! W2 l(R&D).
3 N2 E% y# E+ q( T: Z K x- |Generation of piPS cells1 W) _) a' i8 N) J$ Y
OG2-MEFs were seeded at 5x104 cells per well in a 6-well plate coated with gelatin in normal
/ t- g/ S: W3 A( ?/ A% N& z \MEF media (DMEM supplemented with 10% FBS). On the next day, media was changed to the( |" [& Z) H, c, \: U0 ~
protein transduction media, which were prepared by mixing the recombinant reprogramming2 S) |. _4 J4 ?3 D
proteins at the final concentration of 8 μg/ml with regular mES cell growth medium
) h. F9 u: y# T xsupplemented with 1000 units/ml LIF. After overnight culture in the protein transduction media,
1 r: _3 h/ b+ c' g. ~, G6 ]media was changed to normal mESC growth media, and cells were cultured for additional 366 Y! e3 @4 U6 l% M, Z) T
hours before repeating the same protein transduction cycle. Total four protein transduction
; X ~1 B& Z9 s% ucycles were applied on the cells. After completing protein transduction, cells were then
% C' T7 M5 T e+ l: ]2 e: y# S* mpassaged onto irradiated CF-1 MEF feeder cells at day 9 in normal mESC growth media. Media9 l6 G) p1 R( X3 y9 @' V$ @. }
were changed every 3-4 days until GFP+ colonies were observed around day 30-35. GFP++ `! w% ?) O: j8 t- U" ?( r
colonies were then passaged onto new irradiated MEF feeder cells in normal mESC growth3 [0 G1 q2 s& C
media, and stably maintained and expanded as piPS cells. Some colonies were further selected
1 L& D. C' F% }" g9 `/ jand expanded in the presence of pluripotin (1 μM) or PD0325901 (1 μM). |
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发表于 2012-3-25 09:33
