回复 ghfvcat 的帖子 " D3 b7 R7 a3 w; r8 A% M/ O0 Y7 C4 M! }9 l6 w, ?8 Z
是millipore的3 c7 m5 @' `+ D0 t% {
1. Culture ES cells for five days prior to analyzing AP activity, at low to medium density (NOTE: This 7 f7 X- c6 t |( d; ztime-period is critical if activity levels of AP needs to be observed. According to our findings, five5 G# i, V1 H$ B( R
days of culturing are optimal for good AP stain visualization). + Q- `/ a/ F5 ]) T* |; }2. On day five, aspirate media and fix ES or EC cells with a fixative (e.g. 4% Paraformaldehyde in. H, N% C$ `% Q/ V ]7 g9 W
PBS) for 1-2 minutes., L# v$ D" q) ?1 ~
Note: Do not overfix. Fixing cells longer than 2 minutes will result in the inactivation of alkaline) ?9 h% g$ ?3 w; L
phosphatase.* J3 {6 q6 H7 J$ Z9 n. j: N
3. Aspirate fixative and rinse with 1 X Rinse Buffer. DO NOT allow wells to dry.* r; `: N7 p2 m- t3 g K9 e6 P
, {3 P0 [9 u5 P
4. Prepare reagents for Alkaline Phosphatase staining as described in “Preparation of Reagents”section.' \7 Y& |: m7 C6 u# S$ w
5. Add enough stain solution to cover each well (e.g. 0.5mL for a well of a # L \3 t( C2 Q0 W- |8 T24-well plate). Incubate in dark at room temperature for 15 minutes.% l3 u, |" W. Y x1 Q1 t' [ P
6. Aspirate staining solution and rinse wells with 1 X Rinse Buffer. Cover cells with 1 X PBS to4 I+ k* |5 K9 g E: @
prevent drying and then count the number of colonies expressing AP (red stem cell colonies), 6 G1 x. H m% V* q! i) a1 O- gversus the number of differentiated colonies (colorless).) k6 B$ M- j" F- J$ W
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.作者: ghfvcat 时间: 2011-3-25 16:03
