de Fries R, Mitsuhashi M
Medical Sciences Division, Hitachi Chemical Research Center, Inc., Irvine, California 92715, USA.
J Clin Lab Anal. 1995;9(2):89-95. doi: 10.1002/jcla.1860090203.
Proliferation of human lymphocytes in response to various stimuli has traditionally been assessed by measuring uptake of radiolabeled nucleotides such as 3H-thymidine. We have evaluated a fluorometric assay, which uses the commercially available reagent, alamarBlue, as a potential substitute for the 3H-thymidine assay in measuring proliferation of human lymphocytes. In this assay, alamarBlue is added to a population of cells where it is reduced by mitochondrial enzyme activity. The reduced form of the reagent is fluorescent and can be quantitatively detected. The safety and convenience of the alamarBlue assay make it very attractive for use in the clinical laboratory. In this study peripheral blood mononuclear cells (PBMC) from healthy donors were stimulated using the mitogen Concanavalin A, and proliferation was assessed using either the 3H-thymidine or the alamarBlue assay. The alamarBlue assay reliably detects human PBMC and we found that the linear range of detection was 10(4) cells/well (96-well plate) to 5 x 10(5) cells/well. Detection of human PBMC is highly reproducible and the alamarBlue assay may be suitable in a number of applications where detection or relative quantitation of human PBMC is required. The alamarBlue assay also detected mitogen induced proliferation of PBMC although with a significantly lower level of sensitivity than the standard 3H-thymidine assay.
传统上，人类淋巴细胞对各种刺激的增殖反应是通过测量放射性标记核苷酸（如³H-胸腺嘧啶核苷）的摄取来评估的。我们评估了一种荧光测定法，该方法使用市售试剂alamarBlue，作为³H-胸腺嘧啶核苷测定法在测量人类淋巴细胞增殖中的潜在替代方法。在该测定法中，将alamarBlue添加到细胞群体中，它会被线粒体酶活性还原。试剂的还原形式具有荧光性，可以进行定量检测。alamarBlue测定法的安全性和便利性使其在临床实验室中非常有吸引力。在本研究中，使用促细胞分裂剂刀豆球蛋白A刺激健康供体的外周血单核细胞（PBMC），并使用³H-胸腺嘧啶核苷测定法或alamarBlue测定法评估增殖情况。alamarBlue测定法能够可靠地检测人类PBMC，我们发现检测的线性范围为每孔10⁴个细胞（96孔板）至每孔5×10⁵个细胞。人类PBMC的检测具有高度可重复性，并且alamarBlue测定法可能适用于许多需要检测或相对定量人类PBMC的应用。alamarBlue测定法也检测到了促细胞分裂剂诱导的PBMC增殖，尽管其灵敏度明显低于标准的³H-胸腺嘧啶核苷测定法。
