Department of Urology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.
J Fluoresc. 2010 May;20(3):779-86. doi: 10.1007/s10895-010-0621-5. Epub 2010 Feb 26.
Prerequisites for successful flow cytometry investigations are specific antibodies labeled with appropriate fluorochromes and negligible autofluorescence of the untreated cells at the wavelength of interest. The aim of this study was (a) to characterize frequently used urological carcinoma cell lines with regard to their autofluorescence properties, (b) to demonstrate the autofluorescence as a serious interfering factor on FACS analysis of urological carcinoma cell lines and (c) to suggest an alternative to avoid interfering autofluorescence. Twenty-one cell lines originating from prostate carcinoma, renal cell carcinoma and bladder cancer were included in this study. The various cell lines were read on a flow cytometer in comparison to human erythrocytes as cells with low fluorescence intensity. Urological cell lines show a high autofluorescence when flow cytometry analyses are performed at the frequently used excitation wavelengths at 405 and 488 nm. At excitation wavelength of 633 nm, this problem was reduced and most of the cell lines (14/21) were without autofluorescence at the emission wavelength of 785 nm. In addition, with a spectrofluorometer three exemplary cell lysates were investigated. The above observations were confirmed. The dye APC-Cy7 is one suitable fluorochrome for successful investigation under these measurement conditions.
成功进行流式细胞术研究的前提条件是使用合适的荧光染料标记的特异性抗体，以及在感兴趣的波长处未经处理的细胞的可忽略不计的自发荧光。本研究的目的是(a) 对常用的泌尿上皮癌细胞系的自发荧光特性进行表征，(b) 证明自发荧光是泌尿上皮癌细胞系的 FACS 分析中的一个严重干扰因素，以及 (c) 提出一种替代方法以避免干扰的自发荧光。本研究共纳入了 21 种源自前列腺癌、肾细胞癌和膀胱癌的细胞系。将各种细胞系在流式细胞仪上进行检测，并与人类红细胞进行比较，因为红细胞的荧光强度较低。当在常用的 405nm 和 488nm 激发波长下进行流式细胞术分析时，泌尿上皮细胞系会表现出较高的自发荧光。在 633nm 的激发波长下，这个问题得到了缓解，大多数细胞系（21 种中的 14 种）在 785nm 的发射波长处没有自发荧光。此外，使用分光荧光计对三个典型的细胞裂解物进行了研究。上述观察结果得到了证实。染料 APC-Cy7 是在这些测量条件下成功研究的一种合适的荧光染料。
