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是millipore的
( t' z5 }' {& D* O1. Culture ES cells for five days prior to analyzing AP activity, at low to medium density (NOTE: This
" q0 j8 _. h6 z2 o9 Xtime-period is critical if activity levels of AP needs to be observed. According to our findings, five0 E: z% ?" b1 O: H
days of culturing are optimal for good AP stain visualization).) u5 i9 ~7 R2 |7 @% U7 q) T+ [
2. On day five, aspirate media and fix ES or EC cells with a fixative (e.g. 4% Paraformaldehyde in! q4 g9 i) H( T& u# Q9 F
PBS) for 1-2 minutes.* ?* W; U7 I. W; x; y& k- l3 ~& D
Note: Do not overfix. Fixing cells longer than 2 minutes will result in the inactivation of alkaline$ l/ ^' N+ Y! y8 @8 S
phosphatase.
) `9 F2 D/ ?7 p/ G# v, h8 V/ Y3. Aspirate fixative and rinse with 1 X Rinse Buffer. DO NOT allow wells to dry.; g- E$ a/ z- ^1 y& f+ }
, u# }- h L7 o- w2 x! ?; `% Y4. Prepare reagents for Alkaline Phosphatase staining as described in “Preparation of Reagents”section.
9 l. |% u' _: c. _$ g- D- d5. Add enough stain solution to cover each well (e.g. 0.5mL for a well of a
9 _ h8 X; C: w6 X: h& j24-well plate). Incubate in dark at room temperature for 15 minutes.0 t1 b& l0 j% Z5 ? g1 w* ?$ I3 F
6. Aspirate staining solution and rinse wells with 1 X Rinse Buffer. Cover cells with 1 X PBS to3 e4 @7 o5 U/ M) s& j
prevent drying and then count the number of colonies expressing AP (red stem cell colonies),! Z$ A8 \) Y @ K+ G. m
versus the number of differentiated colonies (colorless).# W7 Z2 R. Q) ?" g
7. AP staining criteria: Greater than 90% of colonies should remain undifferentiated and express alkaline phosphatase in the well containing 103 Units of LIF. P value shall be ³ 0.05. |
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发表于 2011-3-24 17:42
发表于 2011-3-24 18:05
