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使用MEF来源的条件培养基代替feeder。
& H( N2 n" ]/ H" Z# r, g条件培养基制作方法如下：# }: `$ `' y1 a Q" Z6 h8 [9 U
Preparation of MEF- Conditioned Medium (MEF-CM)
: b( b6 O/ t1 _2 a; A* c1. Plate 4 x 106 mitomycin C treated MEFs in a T75 Flask coated with 0.5% gelatin, in complete MEF medium.
5 m: ]- X. k1 {7 }# l) S2. 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.
0 l+ h. d+ T9 K: j8 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.
1 ?' O& y- V, q4 a. |9 S" k" ?2 q& ^4. Add fresh 20% KSR hESC medium containing 4 ng/ml bFGF to the flasks.8 t `, O: J, D# _, t
5. Collect MEF-CM for up to seven days using this procedure.3 O6 C! c4 o4 K6 Z0 |; ^. R: r
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
