MSC成脂成骨诱导图片

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% M3 B! C. R! F# @/ s1uM=1000nM

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回复 stream 的帖子
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干细胞大部分都分化了，有些未分化的也老化了，我用的细胞不是经过流式细胞仪筛选的，有些本身就不是干细胞。

lkzjyx

请问楼主是做的哪种动物的MSC呢？用的什么方法分离和培养的，谢谢   
$ X# ~! G% D  _; d9 U8 }因为我也在做这个方面的研究  我做的是犬的BMSC 想跟你交流下

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5 R5 ]: o9 s0 y% n7 l回复 luoziwei 的帖子" C& {2 l# ?8 O' ^; |) j

2 a, [  n. [" N4 c7 q# C" v6 K7 N4 e很抱歉回复不及时，这几天出门了。3 L# k4 b  H: A5 Q* f
下面是我的染色方法：" t* I, s& x6 i7 j9 |# f
固定前细胞吸去培养基用PBS洗一到二次。& F: Z" l! g! I: L. c3 S
Alizarin Red S staining:( d7 ]! \* t$ w+ b
1. 4%PFA fixation at RT for 30min.
; \/ ]4 }  M! i2.Then wash wish PBS twice.
. J# G8 x$ m! U7 @! Q  A& n1 Z7 ]1 \1 \3. Add 1ml of 2% alizarin red water solution (pH4.1-4.3 use 1% NH4OH adjust pH) stain RT for 20min with gentle shaking on a rocker.6 N& a; w9 q: \0 a/ T% `1 H# X
4.Then wash with PBS for 4 times with gentle shaking. The washing time could be up to 1 hour.' r1 ?6 s* q4 d' g2 R; P( D
5.Leave some PBS in the well and look under microscope.

皮搋子

多少毫升中加1uM？

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& P1 t( R; m6 S" K) U: V$ t1uM=1umol/L 这要取决于你在诱导中的终体积。
' J7 I* |. d; C, e1M=1mol/L

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关于油红O染色步骤：+ \* p/ F2 ^: U$ j7 c2 g
1。 Cell fixation with 4%PFA RT for 30min.
, ?* r& _5 g9 @, }6 P9 H/ k2.   Remove PFA and wash cell with PBS once.
2 Y% g0 a" e" n; }3. Then wash cell with 60% isopropanol for 30 min on a shaker.
9 S/ u$ l0 S4 w: @9 z4. Remove 60% isopropanol and add working oil red O solution stain for 30 min on a shaker.# e& D! K4 v* w0 D. J3 k+ R5 @
5. After 30min remove oil red O stain immediately and wash with PBS for 4 times.
: b. C, @0 j' }6. Leave some PBS in the well and take picture.1 b  [  H; n1 V) u8 c' g$ _/ K1 X
The working oil red solution should be made fresh from the stock each time.
2 N, z5 k8 n9 y' `+ r4 k$ t. GStock solution (175mg oil red O/50ml 100% isopropanol) could store for up to one year.
8 j8 g- x; {4 A  TWorking solution: dilute the oil red O stock solution with PBS(6ml oil red O stock+4ml PBS) filter with filter paper or 0.45um filter unit.

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0 S4 ^' a9 i& Y: r% Uhuman bone marrow MSC

hxf_86

这个图片很好啊~学习了~

luoziwei

回复 Learner 的帖子
( t' z5 e' ]9 `  \. h% j2 j- R
) @1 w8 B$ ~; ^3 H; y有两个小问题想请教一下，你是直接在孔板里诱导染色的？还是把细胞种在玻片上然后取出玻片进行染色拍照的？另外一个问题是，关于油红0染色，用60%的异丙醇洗涤是为什么呢？