Meredith D O, Owen G Rh, ap Gwynn I, Richards R G
Interface Biology, AO Research Institute, Davos-Platz, Switzerland.
Exp Cell Res. 2004 Feb 1;293(1):58-67. doi: 10.1016/j.yexcr.2003.10.005.
The quantification of focal adhesion sites offers an assessable method of measuring cell-substrate adhesion. Such measurement can be hindered by intra-sample variation that may be cell cycle derived. A combination of autoradiography and immunolabelling techniques, for scanning electron microscopy (SEM), were utilised simultaneously to identify both S-phase cells and their focal adhesion sites. Electron-energy 'sectioning' of the sample, by varying the accelerating voltage of the electron beam, combined with backscattered electron (BSE) imaging, allowed for S-phase cell identification in one energy 'plane' image and quantitation of immunogold label in another. As a result, it was possible simultaneously to identify S-phase cells and their immunogold-labelled focal adhesions sites on the same cell. The focal adhesion densities were calculated both for identified S-phase cells and the remaining non-S-phase cells present. The results indicated that the cell cycle phase was a significant factor in determining the density of focal adhesions, with non-S-phase cells showing a larger adhesion density than S-phase cells. Focal adhesion morphology was also seen to correspond to cell cycle phase; with 'dot' adhesions being more prevalent on smaller non-S-phase and the mature 'dash' type on larger S-phase cells. This study demonstrated that when quantitation of focal adhesion sites is required, it is necessary to consider the influence of cell cycle phases on any data collected.
粘着斑位点的定量分析提供了一种可评估的测量细胞-底物粘附的方法。这种测量可能会受到样本内变异的阻碍，这种变异可能源于细胞周期。同时利用放射自显影和免疫标记技术相结合的方法，用于扫描电子显微镜（SEM），以识别S期细胞及其粘着斑位点。通过改变电子束的加速电压对样品进行电子能量“切片”，结合背散射电子（BSE）成像，使得在一个能量“平面”图像中能够识别S期细胞，并在另一个图像中对免疫金标记进行定量分析。结果，可以在同一细胞上同时识别S期细胞及其免疫金标记的粘着斑位点。计算了已识别的S期细胞和存在的其余非S期细胞的粘着斑密度。结果表明，细胞周期阶段是决定粘着斑密度的一个重要因素，非S期细胞的粘附密度比S期细胞大。粘着斑形态也被发现与细胞周期阶段相对应；“点状”粘着在较小的非S期细胞上更为普遍，而成熟的“短划线”型粘着在较大的S期细胞上更为普遍。这项研究表明，如果需要对粘着斑位点进行定量分析，就有必要考虑细胞周期阶段对所收集的任何数据的影响。
