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我把内容放出来，请各位分析一下，谢谢$ D0 k, C+ L6 `! ~ C( X( n
Cell culture
* V2 `( w* y2 s$ S+ E2 OOG2-MEFs were cultured on gelatin-coated dishes in normal MEF media: high-glucose D-MEM, s8 ?7 w# S2 t f: j. q% w) B
(Invitrogen) with 10% FBS, 0.1 mM non-essential amino acids, and 2 mM L-glutamine. piPS
6 V6 W7 a4 F3 scells were cultured on irradiated CF1 MEFs with normal mESC growth media, which consist of2 O, n4 z! y) C# P8 }. A: r* l
Knockout DMEM (Invitrogen) supplemented with 20% KSR, 0.1 mM 2-ME, 2 mM L-glutamine,
5 }# H, q: {5 Q1 [0.1 mM non-essential amino acids, and 1000 units/ml LIF (ESGRO, Chemicon International). The
5 D" W0 m$ `$ ]! {) rpiPS cells were passaged every 3 days as a single cell suspension using 0.05% trypsin/EDTA q- ~7 Q" B( `$ M
and seeded at 1.0x104 cells per cm2 for routine culture. For feeder-free culture, cells are grown' q! N! v& N; k0 G
on gelatin-coated tissue culture dishes in chemically defined media, which consist of Knockout
* s+ Y7 M. R& B. f! tDMEM supplemented with 1xN2, 1xB27, 0.1 mM 2-ME, 2 mM L-glutamine, 0.1 mM nonessential+ c- c5 q R/ U L7 J
amino acids, 50 μg/ml BSA fraction V (GIBCO), 103 units/ml LIF and 10 ng/ml BMP4& `. Z J: }: y; V& _8 V' }
(R&D).
* k1 Y, c% x# y2 PGeneration of piPS cells" I1 G3 O' R. Q1 r4 v) l
OG2-MEFs were seeded at 5x104 cells per well in a 6-well plate coated with gelatin in normal" M6 o$ L$ i# M& K! O
MEF media (DMEM supplemented with 10% FBS). On the next day, media was changed to the: E, q8 g! ?# X @* z7 ]; k O! U
protein transduction media, which were prepared by mixing the recombinant reprogramming
1 W8 e# ~1 Q, Z2 u" oproteins at the final concentration of 8 μg/ml with regular mES cell growth medium
/ o3 h% M. I( A g, nsupplemented with 1000 units/ml LIF. After overnight culture in the protein transduction media,
9 v, E6 D& f5 d( i0 A4 C: fmedia was changed to normal mESC growth media, and cells were cultured for additional 36( ]5 I4 t3 }5 ?8 b7 A3 h
hours before repeating the same protein transduction cycle. Total four protein transduction/ _+ p2 D, j. T* O0 N: N
cycles were applied on the cells. After completing protein transduction, cells were then
1 x0 u7 p; S( T4 I% ]0 d4 N5 Hpassaged onto irradiated CF-1 MEF feeder cells at day 9 in normal mESC growth media. Media
0 W5 q/ k* K5 C0 t0 Swere changed every 3-4 days until GFP+ colonies were observed around day 30-35. GFP+
) V+ ~1 t5 X4 k+ [0 R( S$ `colonies were then passaged onto new irradiated MEF feeder cells in normal mESC growth
; U1 P0 p: L0 U/ emedia, and stably maintained and expanded as piPS cells. Some colonies were further selected
7 s: o# |7 ]8 R' @. vand expanded in the presence of pluripotin (1 μM) or PD0325901 (1 μM). |
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发表于 2012-3-25 09:33
