Papazisis K T, Geromichalos G D, Dimitriadis K A, Kortsaris A H
Theagenion Cancer Institute, Thessaloniki, Greece.
J Immunol Methods. 1997 Oct 27;208(2):151-8. doi: 10.1016/s0022-1759(97)00137-3.
Sulforhodamine B (SRB) protein staining has been widely used for cell proliferation and chemosensitivity testing, substituting for tetrazolium-based assays. However, the cell fixation step in the original assay is subject to error. We tested whether aspiration of medium with an automatic microplate multiwash device prior to fixation improves the method for adherent cells. A panel of adherent cell lines was used. Signal-to-noise ratios were significantly increased in the new assay. Coefficients of variation (CV) between replicate wells were significantly lower especially at lower cell densities. The linearity of the method improved, with absolute linearity over the whole range of cell densities. The aspiration procedure dislodged only negligible numbers of cells. Cytotoxicity testing using the cytotoxic agent paclitaxel showed no IC50 (50% inhibitory concentration) differences between the new and original methods but a better CV was associated with the optimized protocol. We conclude that aspiration of the growth medium prior to fixing comprises a safe and reliable practice which improves CV, linearity and the signal-to-noise ratio of the SRB assay.
磺酰罗丹明B（SRB）蛋白染色已广泛用于细胞增殖和化学敏感性测试，以替代基于四氮唑的检测方法。然而，原始检测方法中的细胞固定步骤容易出错。我们测试了在固定之前使用自动微孔板多洗装置吸出培养基是否能改进贴壁细胞的检测方法。使用了一组贴壁细胞系。在新的检测方法中，信噪比显著提高。重复孔之间的变异系数（CV）显著降低，尤其是在较低细胞密度时。该方法的线性得到改善，在整个细胞密度范围内具有绝对线性。吸出操作仅使极少量细胞脱落。使用细胞毒性药物紫杉醇进行的细胞毒性测试表明，新方法和原始方法之间的IC50（50%抑制浓度）没有差异，但优化后的方案具有更好的CV。我们得出结论，在固定之前吸出生长培养基是一种安全可靠的做法，可改善CV、线性和SRB检测的信噪比。
