Munshi Soumyabrata, Twining Robert C, Dahl Russell
Department of Neuroscience, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
Department of Cellular and Molecular Pharmacology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
J Pharmacol Toxicol Methods. 2014 Sep-Oct;70(2):195-8. doi: 10.1016/j.vascn.2014.06.005. Epub 2014 Jun 13.
The cell viability assay by alamar blue is based on the principle of reduction of the non-fluorescent reagent (resazurin) to a fluorescent compound (resarufin) by the intracellular reducing environment of living cells over time. In the present study, we have for the first time shown that even in the absence of cells, there occurs significant interaction between alamar blue and cell-culture media causing an increase in fluorescence.
We have used Opti-MEM, DMEM and 1:1 DMEM:Opti-MEM as three different media and determined the changes in their relative fluorescence units (RFUs) over time after the addition of 10% (v/v) alamar blue using two-way repeated measures analysis of variance (RM-ANOVA) followed by Tukey's post-hoc test.
Our results show that upon the addition of alamar blue, there occurs a significant increase in RFUs in all the three media over time along with a significantly higher RFU for the Opti-MEM overall (p<0.05). We also show that the time-dependent change in RFU of 1:1 DMEM:Opti-MEM was more gradual compared to that of the other two media.
These findings indicate that the reagent can itself interact with the media causing significantly different fluorescence over time in a manner independent from the effect of intracellular reducing environment of living cells on alamar blue. In addition our results indicate that fluorescence varies as a function of incubation time with the reagent. These findings signify the need for routine subtraction of the background fluorescence of media-only with alamar blue reagent during measurement of cell viability by this method in order to determine an accurate measurement of cell viability.
alamar blue细胞活力测定基于以下原理：随着时间推移，活细胞的细胞内还原环境将非荧光试剂（刃天青）还原为荧光化合物（试卤灵）。在本研究中，我们首次表明，即使在没有细胞的情况下，alamar blue与细胞培养基之间也会发生显著相互作用，导致荧光增加。
我们使用Opti-MEM、DMEM和1:1 DMEM:Opti-MEM作为三种不同的培养基，并在添加10%（v/v）alamar blue后，使用双向重复测量方差分析（RM-ANOVA）及随后的Tukey事后检验，测定它们随时间的相对荧光单位（RFU）变化。
我们的结果表明，添加alamar blue后，所有三种培养基中的RFU均随时间显著增加，总体而言Opti-MEM的RFU显著更高（p<0.05）。我们还表明，与其他两种培养基相比，1:1 DMEM:Opti-MEM的RFU随时间的变化更为平缓。
这些发现表明，该试剂本身可与培养基相互作用，随着时间推移产生显著不同的荧光，其方式独立于活细胞的细胞内还原环境对alamar blue的影响。此外，我们的结果表明，荧光随与该试剂的孵育时间而变化。这些发现表明，在通过该方法测定细胞活力时，需要常规扣除仅含alamar blue试剂的培养基的背景荧光，以便准确测定细胞活力。
