Institute of Biophysics, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
Cell Biol Int. 2010 May 7;34(6):663-8. doi: 10.1042/CBI20090276.
Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V-79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye-exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin-probe MeFASL(10,3) (5-doxylpalmitoyl-methylester), which partitions mainly in cell membranes and the hydrophilic spin-probe TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin-probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin-treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.
细胞分离程序可能会对细胞造成严重损伤。许多研究都需要在测量前将细胞分离，因此，对于那些已经附着在培养皿底部并随后被分离的细胞的研究，当细胞膜的性质发生微小变化（如在膜通透性的光谱研究中）时，实验结果就会出现一个特殊的问题。我们研究了三种不同的分离程序对处于生长平台期（G1 期阻滞）的 V-79 细胞的影响：橡胶刮除法、胰蛋白酶消化法和柠檬酸盐缓冲液处理法。我们通过染料排除试验测量细胞活力；通过电子顺磁共振（EPR）方法使用亲脂性自旋探针 MeFASL(10,3)（5-二氧代棕榈酰甲酯）和水溶性自旋探针 TEMPONE（4-氧代-2,2,6,6-四甲基哌啶-1-氧自由基）测量细胞内的氮氧化物还原动力学和膜流动性，自旋探针主要分布在细胞膜和细胞质中。使用扫描电子显微镜（SEM）观察到由于分离过程导致的细胞损伤。结果发现，胰蛋白酶处理的细胞活力为 91%，柠檬酸盐处理的细胞活力为 85%，橡胶刮除法的细胞活力为 70%。尽管橡胶刮除法对质膜造成了机械损伤，但与其他分离方法相比，膜结构域结构没有明显改变。另一方面，自旋探针还原率既取决于穿过质膜的运输，也取决于细胞的代谢特性，胰蛋白酶法的还原率最高，这表明代谢率受影响最小。只有经过胰蛋白酶处理的细胞在悬浮液中搅拌 4 小时后，还原率保持不变。这些结果表明，与刮细胞或使用柠檬酸盐缓冲液相比，最适合 V-79 细胞的分离方法是用胰蛋白酶分离细胞，并将细胞保持在搅拌的细胞悬浮液中直至测量。这种方法提供了最高的细胞活力，SEM 显微照片上的可见损伤最小，并且细胞的代谢率保持不变。
