Sample Preparation of Adherent Cell Lines for Flow Cytometry: Protocol Optimization-Our Experience with SW-480 colorectal cancer cell line.

The most common method for detection of apoptosis is flow cytometry. In previously published studies there are some uncertainties and problems about the preparation of adherent cell lines for analysis. Thus, the aim of this study is to determine and describe how preparing the sample of SW-480 cells in two different ways affects the reliability of the results. In Protocol 1 the total cell number, cells in flow media and cells which adhere, were used, while in Protocol 2 the medium was removed after cell incubation and only the adherent cells were used. Results show statistically significant changes in percentages of different cell types (viable, apoptosis, and necrosis) between two different protocols. Protocol 2, where the first medium with dead cells were removed and only the cells that were attached to the bottom were used for analysis, give better cell viability in the control sample. Removing the medium is especially recommended for long-term treatments, where the cells consume nutrients and, due to lack, initiate apoptosis. After 72 h, spontaneous apoptosis is detectable in control cells and indicates low viability of the control sample, while in treated cells by proapoptotic substances, together with induced apoptosis leads to cumulative or synergistic effects.