我开始也准备买cck-8的，可1000空要1千多，我最多只做3块96空的板子，太划不来了，找人合买又未凑足10块，只好继续MTT。作者: one 时间: 2012-1-14 12:50
MTT assay for cell proliferation
MTT stock solution: 5mg/ml MTT (Promega) in RPMI-1640 without phenol red.
This solution is filtered through a 0.2 mm filter and stored at 2-8°C.
MTT working solution:
1:10 dilution of the 5mg/ml stock (MTT in RPMI without phenol red).
1. Wash cultured cells with warm RPMI-1640 without phenol red.
2. Prepare MTT working solution.
3. Add MTT working solution into wells being assayed, for example 1.0ml for each well
of 12-well plate. Incubate at 37°C for 30min to 3 hrs (this time depends on cell
density and cell type).
4. At the end of the incubation period, the medium can be moved if working with
attached cells.
5. The converted dye is solubilized with 1ml acidic isopropanol (0.04 M HCl in absolute
isopropanol). Pipette up and down several times to make sure the converted dye
dissolves completely.
6. Transfer the dye solution with the cells into a 1.5 ml eppendorf tube and centrifuge at
13,000 rpm for 2 min.
7. Transfer the supernatant into a new eppendorf tube. Absorbance of the converted dye
is measured at a wavelength of 570nm with background subtraction at 650nm. For the
measurement, use Beckman DU-600 Spectrophotometer and disposable plastic
cuvettes.作者: DNA 时间: 2012-1-14 12:55
我也来说两句：MTT法在用于测定药物的细胞增殖抑制作用上还是有些缺点的，我师兄的一篇文章投到 kidney international （影响因子5.0——6.0吧），结果被退了回来，主要是因为这个问题：
“【Table 2 : the MTT test does not assess proliferation, but only reflects the number of viable cells. The OD at 48 and 72hrs arereduced when compared to .........the authors should perform an additional assay for proliferation assessment ( ex : BrdU or thymidine incorporation ) .The MTT data should be shown as bar graphs, and include a time zero lane (to show if the absolute number of cells has indeed increased at 24hrs or not)】”
以上是退槁原文。师兄先正在做BrdU呢。
所以，我觉得：
正如许多大虾所言，MTT在国外的许多大的药物研发机构中，仍被用于大量药物的粗筛，因为MTT法有太多的优点了：简便、快速、重复性好、价格低、所需细胞少、无放射性等等，在国内更是被大量应用于抗肿瘤药物的筛选极其他方面，MTT的确是个好东东。但是如果想让文章发的更好一点，建议别怕多花钱，用BrdU吧！！！
如果哪位大虾能更详细地介绍一下MTT、BrdU、以及放射性同位素掺入试验等方法的优劣极其原因，那就更好了，谢谢。作者: 月牙牙 时间: 2012-1-14 12:59
