Abstract
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).
Highlights
Flow cytometry is a technique that allows an efficient assessment of multiple cellular parameters at a single cell level in opposition to bulk methods
Fluorescence properties of antibodies that bind to epitopes on the surface of cells can be used in cytometric analysis
We showed the importance of choosing a suitable cell detachment method for the reliability of flow cytometry results
Summary
Flow cytometry is a technique that allows an efficient assessment of multiple cellular parameters at a single cell level in opposition to bulk methods. The technique is a convenient way of assessing the parameters of cells undergoing apoptosis It allows a precise examination of effects evoked by, e.g., anticancer drugs. It allows the study of the expression of the cellular surface proteins, as well as changes in DNA content in cells passing through the cell cycle. Fluorescence properties of antibodies that bind to epitopes on the surface of cells can be used in cytometric analysis. This type of research allows evaluating whether the protein is present on the cell surface. The level of the fluorescence signal allows assessing whether changes in the level of surface protein expression have occurred during cellular analyses compared with available literature data
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