计算溶液所需的质量、体积或浓度。
,适用于对基线干扰要求严格的色谱和分析工作流程。
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在色谱分析、有机合成和交叉偶联反应领域已被 1 篇同行评审文献引用。
产品简介
Alamar Blue检测试剂为细胞增殖和细胞毒性检测提供了一种简便、快速、可靠、安全的方法,适用于高通量检测实验。该检测试剂的主要成分是一种氧化还原指示剂。其在氧化状态下呈现紫蓝色无荧光性,而在还原状态下,转变为呈粉红或红色荧光的还原产物,吸收峰为530-560nm,而发射峰为590nm。
在细胞增殖过程中,细胞内NADPH/NADP、 FADH/FAD、 FMNH/FMN和NADH/NAD的比值升高,处于还原环境。摄入细胞内的染料被这些代谢中间体及细胞色素类还原后释放到细胞外并溶于培养基中,使培养基从无荧光的靛青蓝变成有荧光的粉红色。最后用普通分光光度计或荧光光度计进行检测,吸光度和荧光强度与活性细胞数成正比。
操作说明
1. 在100µl细胞悬液中加入10µl的检测试剂,在细胞培养箱内孵育2-6小时,培养基的颜色由靛青蓝开始变成粉红色就可进入下一步。
2. 推荐使用荧光酶标仪进行检测,激发光波长在530-560 nm之间,发射光波长为590 nm,记录相对荧光单位(RFU)。
3. 绘制标准曲线或细胞生长曲线:纵座标(Y 轴)为相对荧光单位(RFU);横坐标(X 轴)为细胞数或时间点或药物浓度。
注意事项
1. 合适密度的细胞可以增加检测灵敏度。对于96孔板,我们建议每孔接种100微升细胞,细胞浓度范围为:贴壁细胞在100-10,000/孔,悬浮细胞在2,000-50,000/孔,并以培养基为空白对照。对于384孔板,细胞浓度和接种量均减半。
2. 整个过程均应为无菌操作,因为微生物污染物同样可以还原检测试剂而影响实验结果。
3. 注意接种细胞浓度和加入检测试剂后孵育时间。细胞浓度过高或孵育时间过长,会导致继发性还原反应,产生无色和荧光消失。
4. 孵育时,须避光。
5. 本产品可以使用荧光或分光光度检测,但荧光的灵敏度高,实验误差小,推荐使用荧光检测。
Product Introduction
Alamar Blue detection reagent provides a simple, rapid, reliable and safe method for cell proliferation and cytotoxicity detection, which is suitable for high-throughput detection experiments. The main component of the detection reagent is a redox indicator. In the oxidized state, it appears purple-blue and non-fluorescent, while in the reduced state, it turns into a reduction product with pink or red fluorescence, with an absorption peak of 530-560nm and an emission peak of 590nm.
In the process of cell proliferation, the ratios of NADPH/NADP, FADH/FAD, FMNH/FMN and NADH/NAD in the cell increase and are in a reducing environment. The dye taken into the cell is reduced by these metabolic intermediates and cytochromes and then released outside the cell and dissolved in the culture medium, changing the culture medium from non-fluorescent indigo blue to fluorescent pink. Finally, use an ordinary spectrophotometer or fluorophotometer for detection, and the absorbance and fluorescence intensity are proportional to the number of active cells.
Instructions
1. Add 10µl of detection reagent to 100µl of cell suspension, and incubate in a cell incubator for 2-6 hours. The color of the medium changes from indigo blue to pink and you can proceed to the next step.
2. It is recommended to use a fluorescence microplate reader for detection, the excitation light wavelength is between 530-560 nm, the emission light wavelength is 590 nm, and the relative fluorescence unit (RFU) is recorded.
3. Draw a standard curve or cell growth curve: the ordinate (Y axis) is the relative fluorescence unit (RFU); the abscissa (X axis) is the cell number or time point or drug concentration.
Precautions
1. The appropriate density of cells can increase the detection sensitivity. For 96-well plates, we recommend seeding 100 microliters of cells per well. The cell concentration range is: 100-10,000/well for adherent cells, 2,000-50,000/well for suspension cells, and medium as a blank control. For 384-well plates, the cell concentration and seeding volume are both halved.
2. The whole process should be aseptic operation, because microbial contaminants can also reduce the detection reagents and affect the experimental results.
3. Pay attention to the concentration of inoculated cells and the incubation time after adding detection reagents. If the cell concentration is too high or the incubation time is too long, it will cause a secondary reduction reaction, resulting in colorlessness and disappearance of fluorescence.
4. When incubating, avoid light.
5. This product can use fluorescence or spectrophotometric detection, but the sensitivity of fluorescence is high, and the experimental error is small. Fluorescence detection is recommended.