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使用MEF来源的条件培养基代替feeder。, K6 J$ l4 Z. }' k" [
条件培养基制作方法如下：
* m$ W* X+ W+ \Preparation of MEF- Conditioned Medium (MEF-CM)8 b0 ^' Z) Y" B2 J I, e3 G6 {
1. Plate 4 x 106 mitomycin C treated MEFs in a T75 Flask coated with 0.5% gelatin, in complete MEF medium.
2 T3 V2 N! @! r# g5 {8 `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.
/ b/ X8 @4 k# z$ @) b5 I: E- e4 |3. 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.
7 ^1 s6 M9 Y l/ k, D" i4. Add fresh 20% KSR hESC medium containing 4 ng/ml bFGF to the flasks.
3 H; Z3 e' R# A5. Collect MEF-CM for up to seven days using this procedure.. Y7 U7 u* j$ H$ Q- J
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
