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方法指南：细胞增殖和细胞活性检测方法指南（点击查看/下载）PDF
实验方案
描述
Trypan Blue Solution, 0.4%, is routinely used as a cell stain to assess cell viability using the dye exclusion test. This test is often performed while counting cells with the hemocytometer during routine subculturing, but can be performed any time cell viability needs to be determined quickly and accurately. The dye exclusion test is based upon the concept that viable cells do not take up impermeable dyes (like Trypan Blue), but dead cells are permeable and take up the dye.实验方案
The following procedure will enable you to accurately determine the cell viability. Cell viability is calculated as the number of viable cells divided by the total number of cells within the grids on the hemacytometer. If cells take up trypan blue, they are considered non-viable.
% viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100
To calculate the number of viable cells per mL of culture, use the formula below. Remember to correct for the dilution factor
- Determine the cell density of your cell line suspension using a hemacytometer.
- Prepare a 0.4% solution of trypan blue in buffered isotonic salt solution, pH 7.2 to 7.3 (i.e., phosphate-buffered saline).
- Add 0.1 mL of trypan blue stock solution to 1 mL of cells.
- Load a hemacytometer and examine immediately under a microscope at low magnification.
- Count the number of blue staining cells and the number of total cells. Cell viability should be at least 95% for healthy log-phase cultures.
% viable cells = [1.00 – (Number of blue cells ÷ Number of total cells)] × 100
To calculate the number of viable cells per mL of culture, use the formula below. Remember to correct for the dilution factor
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中英文名称：台盼蓝（Trypan Blue）或称台盼兰、锥虫蓝
CAS号：72-57-1
分子式：C34H24N6O14S4Na4
分子量: 960.82
可溶于水（10mg/ml）
是细胞活性染料，常用于检测细胞膜的完整性。还常用于细胞是否存活。活细胞不会被染成蓝色，而死细胞会被染成淡蓝色。
台盼蓝可被巨噬细胞吞噬，故可用于巨噬细胞的活体染色剂。
正常的活细胞，胞膜结构完整，能够排斥台盼蓝，使之不能够进入胞内；而丧失活性或细胞膜不完整的细胞，胞膜的通透性增加，可被台盼蓝染成蓝色。通常认为细胞膜完整性丧失，即可认为细胞已经死亡，这与中性红作用相反。因此，借助台盼蓝染色可以非常简便、快速地区分活细胞和死细胞。台盼蓝是组织和细胞培养中最常用的死细胞鉴定染色方法之一。注意凋亡小体也有台盼蓝拒染现象。
台盼蓝染色后，通过显微镜下直接计数或显微镜下拍照后计数，就可以对细胞存活率进行比较精确的定量。
台盼蓝染色只需3-5分钟即可完成，并且操作非常简单。
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步骤
1、胰酶消化贴壁细胞，制备单细胞悬液，并作适当稀释。
2、染色：细胞悬液与0.4%台盼蓝溶液以9:1混合混匀。（终浓度0.04%）
3、计数：在三分钟内，分别计数活细胞和死细胞。 1 e7 e* I* S） ^% X8 U$ K- _
4、镜下观察，死细胞被染成明显的蓝色，而活细胞拒染呈无色透明状。
5、统计细胞活力：活细胞率（%）= 活细胞总数/（活细胞总数+死细胞总数）×100%
