Huang Kuang Tzu, Chen Yen Hao, Walker Ameae M
University of California, Riverside, CA 92521-0121, USA.
Biotechniques. 2004 Sep;37(3):406, 408, 410-2. doi: 10.2144/04373ST05.
Soluble formazan assays are widely used for cell number assessment. However, in our hands, we observed frequent occasions in which the actual cell number was at odds with the assay reading. In this study, we have determined that (i) a large proportion of the reading obtained in commonly used culture media can be caused by media component amplification of formazan production in a way that cannot be corrected for by media-only controls; (ii) the albumin present in 10% serum can reduce the assay absorbance by 50% so that an actual doubling of cell number can be obscured; and (iii) this latter effect is dependent on the concentration of fatty acids. To counter these problems, we have developed a protocol that gives consistent readings that are fully representative of cell number while retaining some of the original advantages of soluble formazan assays.
可溶性甲臜测定法被广泛用于细胞数量评估。然而，在我们的实验中，我们经常观察到实际细胞数量与测定读数不一致的情况。在本研究中，我们确定：（i）常用培养基中获得的大部分读数可能是由于培养基成分以无法通过仅含培养基的对照校正的方式放大了甲臜的产生；（ii）10%血清中存在的白蛋白可使测定吸光度降低50%，从而可能掩盖细胞数量实际的翻倍情况；（iii）后一种效应取决于脂肪酸的浓度。为解决这些问题，我们开发了一种方案，该方案能给出与细胞数量完全相符的一致读数，同时保留了可溶性甲臜测定法的一些原有优点。
