Koníková E, Babusíková O, Kusenda J, Glasová M
Cancer Research Institute, Slovak Academy of Sciences, Bratislava.
Neoplasma. 1992;39(6):337-42.
The cells of some human leukemia-lymphoma T cell lines (JURKAT, MOLT4), B cell lines (DAUDI, U-266) and of myeloid U-937 cell line were characterized for their surface membrane and cytoplasmic marker profiles. The usefulness of some fixation and permeabilization methods of cell membrane for detection of cytoplasmic markers by flow cytometry was studied. The methods of cell fixation in suspension were found to be more sensitive than the methods of cell fixation in smears. With the very short buffered formaldehyde-acetone (BFA) fixation used in this study an optimal penetration of the monoclonal antibodies (MoAbs) through the plasma membrane and specific binding to the appropriate structures were achieved. CD22 antigen was detected in cytoplasm but not on membrane of DAUDI cells. In another B cell line, U-266, CD22 antigen was present both in cell membrane and cytoplasm. The marker corresponding to anti-CD19 MoAb was detected in cytoplasm but was absent on membrane of U-266 cells. Furthermore, the antigen estimated by anti-CD3 MoAb could be detected intracellularly in cells of both T cell lines tested, while it was absent on cell membrane of these cells. The phenotypic study of U-937 cells showed that the majority of cells expressed myeloid associated antigens. In our study the CD14 marker detected on cell surface membrane of U-937 cells was missing in their cytoplasm. The surface antigens remained intact after BFA fixation enabling a simultaneous detection of membrane and cytoplasmic markers in double immunofluorescence studies. Through this combination of markers minor cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for a rapid and quantitative immunodiagnosis.
对一些人类白血病 - 淋巴瘤T细胞系（JURKAT、MOLT4）、B细胞系（DAUDI、U - 266）以及髓系U - 937细胞系的细胞进行了表面膜和细胞质标志物谱的特征分析。研究了一些细胞膜固定和通透方法在通过流式细胞术检测细胞质标志物方面的实用性。发现悬浮细胞固定方法比涂片细胞固定方法更灵敏。在本研究中使用的极短时间的缓冲甲醛 - 丙酮（BFA）固定法，可使单克隆抗体（MoAbs）最佳地穿透质膜并与相应结构特异性结合。在DAUDI细胞的细胞质中检测到CD22抗原，但在细胞膜上未检测到。在另一个B细胞系U - 266中，CD22抗原同时存在于细胞膜和细胞质中。与抗CD19 MoAb对应的标志物在U - 266细胞的细胞质中被检测到，但在细胞膜上不存在。此外，抗CD3 MoAb估计的抗原在两种测试的T细胞系的细胞内均可检测到，而在这些细胞的细胞膜上不存在。U - 937细胞的表型研究表明，大多数细胞表达髓系相关抗原。在我们的研究中，在U - 937细胞表面膜上检测到的CD14标志物在其细胞质中缺失。BFA固定后表面抗原保持完整，使得在双重免疫荧光研究中能够同时检测膜和细胞质标志物。通过这种标志物组合，可以检测到少量细胞群体。人类造血细胞系可作为快速定量免疫诊断的可靠模型系统。
