Kim H M, Han S B, Kim M S, Kang J S, Oh G T, Hong D H
Korea Research Institute of Bioscience and Biotechnology, KIST, Yusung, Taejon City, Korea.
J Pharmacol Toxicol Methods. 1996 Nov;36(3):163-9. doi: 10.1016/s1056-8719(96)00113-x.
The fixation procedures in sulforhodamine B (SRB) assay for human leukemia cells were modified to produce more reliable results. It was found that the concentration of the fixative agent, trichloroacetic acid (TCA), was critical in the selective fixation of cellular protein. While a TCA solution of 80% fixed both cells and serum proteins, a 50% solution fixed only cells with a very low interference of the serum proteins. Accordingly, we selected 50% TCA as a fixative agent which made the final absorbance of the SRB assay to be exactly matched to the cell density with a small deviation and a low background. Besides the change of TCA concentration, a precentifugation of microplate just before fixation also improved the previous assay procedures in the two points of view. The 2-h standing step was simply substituted for only 1 min of centrifugation. Both the rapid and slow application of TCA solution in fixation produced the same extents of fixation. In an actual application, these two kinds of modifications in the previous SRB assay procedure were also proved to be effective in the determination of cytotoxicities of doxorubicin by using human leukemias.
对用于人类白血病细胞的磺酰罗丹明B（SRB）检测中的固定程序进行了改进，以产生更可靠的结果。发现固定剂三氯乙酸（TCA）的浓度对于细胞蛋白的选择性固定至关重要。80%的TCA溶液既能固定细胞蛋白也能固定血清蛋白，而50%的溶液仅固定细胞，对血清蛋白的干扰非常低。因此，我们选择50%的TCA作为固定剂，这使得SRB检测的最终吸光度与细胞密度精确匹配，偏差小且背景低。除了改变TCA浓度外，在固定前对微孔板进行离心从两个角度改进了先前的检测程序。将2小时的静置步骤简单地替换为仅1分钟的离心。在固定过程中快速和缓慢加入TCA溶液产生的固定程度相同。在实际应用中，先前SRB检测程序中的这两种改进在用人白血病细胞测定阿霉素的细胞毒性时也被证明是有效的。
