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使用MEF来源的条件培养基代替feeder。% `4 f) T( z+ P0 A0 M* {
条件培养基制作方法如下：* Z% |+ Q; |! ]* g5 w
Preparation of MEF- Conditioned Medium (MEF-CM)7 r1 a) X/ s6 _* X. {; D0 s5 E
1. Plate 4 x 106 mitomycin C treated MEFs in a T75 Flask coated with 0.5% gelatin, in complete MEF medium.
% ?/ I! X+ n1 ^9 A! i2 ?2. The following day, replace the MEF medium with 37.5 ml 20% KSR hESC medium containing 4 ng/ml bFGF, and incubate for 24 hrs at 37°C, 5% CO2.
. j; `. Q/ U2 M6 y$ k+ I5 j3. Collect MEF-CM from the flasks after 24 hrs and 0.22 μM filter sterilize. MEF-CM can be used fresh or can be frozen.) T J1 q% P' A" `$ ^3 s
4. Add fresh 20% KSR hESC medium containing 4 ng/ml bFGF to the flasks.+ U9 K2 \/ I5 B7 F& o+ c9 T/ E
5. Collect MEF-CM for up to seven days using this procedure.) Z a( o+ g6 H
6. Depletion of L-Glutamine and bFGF from the MEF-CM is assumed, and L-Glutamine (to 2 mM final), and bFGF (to 4 ng/ml final) are therefore added back prior to using MEF-CM with hESCs. Freshly thawed ß-Mercaptoethanol (ß-Me) is added to 0.1 mM final fresh each day of use. |
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发表于 2010-3-17 17:05
发表于 2010-3-17 17:24
