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[求助] 材料表面細(xì)胞活性怎么衡量

各位兄弟姐妹們，（之前發(fā)信問了幾個(gè)牛人同學(xué)，都沒有回，無意冒犯，但是這個(gè)問題還比較急，現(xiàn)在卡著我，所以又發(fā)帖來咨詢一下）請(qǐng)教一下關(guān)于生物細(xì)胞試驗(yàn)的一個(gè)問題，(非生物專業(yè)，只在網(wǎng)上搜索過MTT相關(guān)內(nèi)容）望不吝賜教。

我的目標(biāo)是觀測一種材料表面形態(tài)對(duì)細(xì)胞的影響。初步設(shè)計(jì)的試驗(yàn)方案是在實(shí)驗(yàn)組和對(duì)照組材料上面分別培養(yǎng)細(xì)胞，一定時(shí)間后（24h,48h,72h）對(duì)比兩邊細(xì)胞的差異。
現(xiàn)在的問題是由于不是生物專業(yè)，不知道用什么方法來對(duì)比兩組細(xì)胞，甚至不知道培養(yǎng)何種細(xì)胞比較好。我目前的想法是 1. 用電鏡直接觀看細(xì)胞形態(tài)變化 /粘附 2. 測量活細(xì)胞密度（不知道怎么測） 3. MTT 分析細(xì)胞活性
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1樓 2013-01-24 00:38:21

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紅舟流星: 金幣+5, BM-EPI+1, http://emuch.net/bbs/viewthread.php?tid=5464228投票通過~ 2013-01-26 17:33:55

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10樓: Originally posted by bbhgchina at 2013-01-24 19:36:19
Actually I am planning to bring down the surface topography to nano meter scale, to explore the details how surface structure affect cells.

I have read couple dozens of articles, they observed th ...

sounds interesting! wish u good luck.

you'd better have more discussion with biologists, especially with a developmental biology background or cell biology, to design a suitable but valuable model to study (cell type, influencing factor...)

Here is one about nanotopographic influences:

1. McMurray, R. J.; Gadegaard, N.; Tsimbouri, P. M.; Burgess, K. V.; McNamara, L. E.; Tare, R.; Murawski, K.; Kingham, E.; Oreffo, R. O. C.; Dalby, M. J., Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency. Nat Mater 2011, 10, (8), 637-644.

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11樓2013-01-24 21:01:26

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好像問題太模糊了。再增加點(diǎn)描述

想對(duì)比經(jīng)過處理的材料表面形態(tài)，比如說表面溝槽，洞，突起等等對(duì)細(xì)胞的增值，粘附，活性的影響；對(duì)照組就是一個(gè) ‘平板’

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2樓2013-01-24 00:45:39

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★ ★
感謝參與，應(yīng)助指數(shù) +1
紅舟流星: 金幣+2, 專家考核, 歡迎來到生物功能材料板塊積極應(yīng)助交流 2013-01-24 04:34:02

ATP analysis could be used to measured the loss in viability because a decrease in ATP production can be linked
to a decrease in cell number.

Specifically, ATP could be quantified by measuring the light produced through ATP's reaction with a naturally occurring firefly enzyme-Luciferase using a luminometer. The amount of light produced corresponds proportionally to the amount of live cells present in the sample.

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3樓2013-01-24 01:03:24

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★
markar: 金幣+1, 應(yīng)助指數(shù)+1, 感謝回帖交流！ 2013-01-25 12:23:20

Are u using multipotent cells like hMSCs?
if yes, you could try high-content imaging (see the proliferation) and immunofluorescence Staining (see the expression of alkaline phosphatase (ALP), a marker for early osteogenic differentiation)

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4樓2013-01-24 01:55:37

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引用回帖:

3樓: Originally posted by aircraftvip at 2013-01-24 01:03:24
ATP analysis could be used to measured the loss in viability because a decrease in ATP production can be linked
to a decrease in cell number.

Specifically, ATP could be quantified by measuring t ...

Thank you sir. So the name of this sort of analysis is ' the ATP analysis‘?
Are these reactions you mentioned?
luciferin + ATP → luciferyl adenylate + PPi
luciferyl adenylate + O2 → oxyluciferin + AMP + light
But, are these reaction happening in every type of cell like fibroblasts and epithelials, which are likely to be used in my experiments according to articles in this field.

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5樓2013-01-24 02:18:57

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4樓: Originally posted by aircraftvip at 2013-01-24 01:55:37
Are u using multipotent cells like hMSCs?
if yes, you could try high-content imaging (see the proliferation) and immunofluorescence Staining (see the expression of alkaline phosphatase (ALP), a mark ...

Thank you sir, this makes more sense to me now.

To be honest,at this stage, I still have not decided yet which type of cell to use. I will probably go for fibroblasts or epithelials.

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6樓2013-01-24 02:25:28

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6樓: Originally posted by bbhgchina at 2013-01-24 02:25:28
Thank you sir, this makes more sense to me now.

To be honest,at this stage, I still have not decided yet which type of cell to use. I will probably go for fibroblasts or epithelials....

Why fibroblasts?

For your study, the influence of geometry on cell fate, only multipotent stem cells are applicable. Otherwise, the influence might not be so significant.

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7樓2013-01-24 02:52:11

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7樓: Originally posted by aircraftvip at 2013-01-24 02:52:11
Why fibroblasts?

For your study, the influence of geometry on cell fate, only multipotent stem cells are applicable. Otherwise, the influence might not be so significant....

May I ask why ' only multipotent stem cells are applicable' ?
Sorry if this is too simple, I'm not a biology majored.

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8樓2013-01-24 03:41:11

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★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★
紅舟流星: 金幣+1, 應(yīng)助指數(shù)+1, 專家考核, 歡迎來到生物功能材料板塊積極應(yīng)助交流 2013-01-24 12:43:09
bbhgchina: 金幣+10, ★★★很有幫助 2013-01-24 19:19:01

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8樓: Originally posted by bbhgchina at 2013-01-24 03:41:11
May I ask why ' only multipotent stem cells are applicable' ?
Sorry if this is too simple, I'm not a biology majored....

during differentiation, multipotent stem cells could read their microenvironmental signals, integrate them intracellularly, and make varying decisions accordingly. E.g., mesenchymal stem cells, could differentiate into both fibroblasts and osteoblasts according to the matrix stiffness. These points help to explain the development of different tissues.

Question:
why do u perform these experiments?
what is your novelty of your work?
have u read some relevant literatures on this topic?

As I know, it is generally not new. See the former work of Christopher Chen and Dennis Discher:

Geometric Control of Cell Life and Death
Science 30 May 1997:
Vol. 276 no. 5317 pp. 1425-1428

D.E. Discher, P. Janmey, Y-L. Wang. Tissue cells feel and respond to the stiffness of their substrate. Science 310:1139-1143 (2005).

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9樓2013-01-24 08:21:32

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9樓: Originally posted by aircraftvip at 2013-01-24 08:21:32
during differentiation, multipotent stem cells could read their microenvironmental signals, integrate them intracellularly, and make varying decisions accordingly. E.g., mesenchymal stem cells, coul ...

Actually I am planning to bring down the surface topography to nano meter scale, to explore the details how surface structure affect cells.

I have read couple dozens of articles, they observed the cells' attachment and proliferation on certain nano-patterns, but I have not looked into how surface structure affect stem cell differentiation.

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10樓2013-01-24 19:36:19

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