Impact of cell harvesting methods on detection of cell surface proteins and apoptotic markers

Assays based on the flow cytometry technique allow a convenient analysis of multiple cellular parameters; however, their results should be interpreted cautiously due to a strong impact of confounding factors. Different techniques in cell culturing such as either enzymatic or mechanic detachment of adherent cells can heavily influence the structure of the cell membrane or presence of the surface antigens leading to strong false positive signals, and finally, substantial experimental bias. The aim of our study was to assess and compare the impact of cell harvesting methods (both enzymatic and non-enzymatic) on the apoptosis process and on the surface antigen cytometric analyses. We found significant differences in the quality of analysis in terms of the amount of detected surface markers determined by the detachment method. Our results demonstrated clearly how important it is to carefully choose the appropriate detachment method and may help to avoid mistakes in experiment planning. In conclusion, we recommend to adjust the detachment method to the type of analyzed markers (surface antigens or translocated phosphatidylserine).

基于流式细胞术（flow cytometry）的检测方法可便捷开展多项细胞参数的分析，但由于混杂因素的影响较为显著，对其检测结果的解读需格外谨慎。贴壁细胞的酶解或机械解离等不同细胞培养处理方式，会显著改变细胞膜结构或影响表面抗原的表达，进而产生强烈的假阳性信号，最终导致显著的实验偏倚。本研究旨在评估并比较细胞收获方法（涵盖酶解法与非酶解法）对细胞凋亡（apoptosis）过程及表面抗原流式细胞分析的影响。我们发现，不同解离方法下所检测到的表面标记物数量存在显著的分析质量差异。本研究结果清晰阐明，审慎选择合适的细胞解离方法至关重要，同时可为规避实验规划中的失误提供参考。综上，我们建议根据待分析标记物的类型（表面抗原或易位的磷脂酰丝氨酸（phosphatidylserine））调整对应的细胞解离方法。