询问一个关于基质胶的问题！

ahfjwangrui

各位专家们，大家好，做肿瘤干细胞，我看到文献上说可以用ECM gel 铺板培养，我今天询问了这个胶，但不知道具体用法和使用范围及意义，请有用过这玩意的专家给个答案，多谢！

胡问成

回复 1# ahfjwangrui
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( j$ u: g5 R8 P# U2 I    看你要用肿瘤干细胞做什么，基质胶一般用来检测细胞的侵袭、生长能力，如果你要做肿瘤干细胞的克隆、生长能力可以用软琼脂糖克隆实验，不过比较麻烦些

ahfjwangrui

回复 2# 胡问成
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    我是看很多文献说是在培养干细胞时铺板用

shihuijunm

Application：Recommended for use as a cell culture substratum. For a 24-well plate, use 230-250 μl/well. For a 96-well plate, use 50-100 μl/well. Thaw gel overnight at 2-8 °C before use. The thawed gel may be diluted up to two-fold with cold (2-8 °C) Dulbecco′s Modified Eagle′s Medium. Gel dilutions should be made before it is added to the plate. ECM will gel within 5 minutes at 20 °C. For prolonged manipulations, work should be conducted below 10 °C. Dispense gel to wells of a multiwell plate using pipettes pre-cooled to 2-8 °C. A gel forms at 37 °C and maintains this form with culture medium for at least 14 days. Cells may be plated on top of a thin gel layer (0.5 mm) or cultured inside a 1 mm layer. When cultured inside, cells should be added to the gel prior to plating at a recommended density of 3-4 × 104 cells per mL. To dissociate cells from the gel, use protease (dispase) dissolved in PBS without calcium, magnesium, and EDTA at a working concentration of 0.6-2.4 units/ml. " @0 }2 C$ U  Y/ m8 }! V
  Epithelial cells, endothelial cells, muscle cells, nerve cells, tumor cells
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5 A3 r& H* G( y+ z" I2 ^, F! wCaution：ECM gel may be stored up to 72 hours at 2-8 °C.
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Other Notes：ECM gel is composed primarily of laminin, collagen type IV, heparan sulfate proteoglycan and entactin. Approximately 8-12 mg/ml basement membrane matrix protein in Dulbecco′s modified Eagle′s medium with 50 μg/ml gentamicin.6 B# c; M5 E7 Q8 u  @! x% x
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Properties
7 a6 u  Y% r$ E5 psterility dialyzed against chloroform / @9 l/ }4 r+ W  ]0 J& t1 g
form liquid - Z  s" {1 ?0 t- k6 s. E# |4 k
concentration 8 - 12 mg/mL
1 E+ o. R; m. K& K8 P; [surface coverage 6‑10 μg/cm2 0 `( Z4 m2 ?! w; H( p1 r  A
total impurities endotoxin, tested
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5 R$ q+ F3 ]8 r7 Ishipped in dry ice 3 g- w4 V9 F3 p# p
storage temp. −20°C

hyx5415

楼主用基质胶养肿瘤干细胞结果怎么样？CSC能长起来么，会不会分化？

leiwang

我用含生长因子的ECM培养CSCs，in matrigel 会分化，on matrilgel 会出现类似于tubeformation的结果，应该也是一种分化现象。

hyx5415

回复 leiwang 的帖子
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  }5 R' \' G6 c8 [* H这个分化和生长因子不知道关系大不大，还是matrigel本身基本成分促进了分化？

hyx5415

回复 leiwang 的帖子
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5 k" p$ w: r) \* q% M还有请问你培养的时候是无血清培养还是含血清培养？谢谢！

leiwang

生长因子应该很重要，我培养的时候是完全培养基含血清的，就是为了做分化的。

leiwang

忘记说了，有一次我用BD不含生长因子的ECM做分化，效果差很多。生长因子在这里的作用应该是比较重要的。