Ostrowski K, Watson J V, Barnard P J, Barnard E A, Thomas K, Freedman L, de Stavola B
Histochemistry. 1986;85(5):423-9. doi: 10.1007/BF00982673.
A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These "free" cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer. A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from inaccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%-10%.(ABSTRACT TRUNCATED AT 250 WORDS)
研究细胞与底物相互作用的一种常用方法是测量细胞与不同底物或培养细胞层的附着情况。附着情况的评估方法有两种，一种是通过闪烁计数法对预先标记的黏附细胞进行计数，另一种是通过光学显微镜观察细胞形态，有时还会借助自动图像分析技术进行细化评估。这些方法存在诸多缺点。本文建议使用荧光激活流式细胞光度法（FC），该方法可直接对未黏附细胞进行计数。在黏附实验完成后，将这些“游离”细胞从微量滴定板孔中去除。然后加入荧光聚苯乙烯珠形式的内标，以便评估黏附在孔壁上的细胞百分比。流式细胞光度法随后基于对大量细胞的分析生成数据。通过对计数细胞和计数荧光珠进行明确区分，可消除计数误差。结果可由计算机在线处理。使用小鼠脾细胞悬液评估该方法因移液不准确、戊二醛对溴化乙锭（EB）染色的干扰以及仪器误差而产生的总体误差。每次黏附实验均通过染色和后固定终止，结果表明，与未固定的原始细胞相比，这不会对细胞计数产生影响。然而，预固定会淬灭EB染色，并会干扰计数过程。该技术的总体标准误差为5%-10%。（摘要截取自250字）
