MSC成脂成骨诱导图片

Learner

回复 皮搋子 的帖子9 S* s( y  v6 V3 p9 e
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1uM=1000nM

Learner

回复 stream 的帖子( `4 U' H; f" ?% i* C

3 B* D, a& c/ q9 ?  q% i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   " p$ I" Z) O8 o5 e6 n, C
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

Learner

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( Z% x/ i: l, e9 m, a& x" d7 a& ?/ @回复 luoziwei 的帖子9 a9 }1 f3 n$ F8 f7 f9 z$ ]
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很抱歉回复不及时，这几天出门了。
' o' U+ c2 ]6 D; B( A( D下面是我的染色方法：3 K* h' O% I7 c7 E! ~! a
固定前细胞吸去培养基用PBS洗一到二次。
) ^2 ?7 O/ }, [: R& j4 pAlizarin Red S staining:
+ ~! B0 T( }3 \1. 4%PFA fixation at RT for 30min.
) o7 x9 o9 v+ o- o7 ~, o2.Then wash wish PBS twice.
6 M4 z7 h2 t2 |9 L6 Z3. 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.% B3 ^4 @2 ^! T; g0 }* V( Z, a
4.Then wash with PBS for 4 times with gentle shaking. The washing time could be up to 1 hour.5 v9 F6 \& ?0 O, k# A7 V
5.Leave some PBS in the well and look under microscope.

皮搋子

多少毫升中加1uM？

Learner

回复 皮搋子 的帖子$ U" V- M0 g! \- ~( N- Q
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1uM=1umol/L 这要取决于你在诱导中的终体积。( A& C. d7 ^  ]7 b$ _
1M=1mol/L

Learner

回复 luoziwei 的帖子2 l6 _, \+ H7 a# e3 G
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关于油红O染色步骤：
0 B! Y* H( Z# x1 `, C1。 Cell fixation with 4%PFA RT for 30min.
2 X( B" ~  ?; h, v2.   Remove PFA and wash cell with PBS once.
( _$ K; {, X7 |. H$ u3. Then wash cell with 60% isopropanol for 30 min on a shaker.
$ b, u# h) z. {1 \9 D, W4. Remove 60% isopropanol and add working oil red O solution stain for 30 min on a shaker.! a2 X( a- n8 R3 O6 ]2 C& ~: ?
5. After 30min remove oil red O stain immediately and wash with PBS for 4 times./ M7 h# {) x% u+ N2 w" {$ c  L
6. Leave some PBS in the well and take picture.8 Y6 C! P0 q1 {: A3 z( q2 H
The working oil red solution should be made fresh from the stock each time.
3 m, ^: ?2 D2 v: M: |1 AStock solution (175mg oil red O/50ml 100% isopropanol) could store for up to one year.
* }3 U& v& {, D$ T7 g( [0 H' X9 rWorking 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.

Learner

回复 lkzjyx 的帖子
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- |% z: s. w" F/ E: ?% S6 lhuman bone marrow MSC

hxf_86

这个图片很好啊~学习了~

luoziwei

回复 Learner 的帖子
3 G' A: |$ y" P1 [8 {/ a. R5 i* k) a
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？