Data Availability StatementAvailable upon request. concentrations between 0.1 and 4%. After total PFA fixation, cell surface fluctuation decreased to 71% of live cell, while the Youngs modulus increased by fivefold compared to that of live cells. These results provide a deeper understanding of how cells react to chemical treatment with PFA that takes into account not only the traditional chemical understanding of PFAs effect upon the cell, but now also the cells surface-based mechanical properties that were targeted in this study. It is now apparent that PFA fixation enables the opening of distributed proteins across the cell surface, a critical process that facilitates common crosslinking. Cell membranes that are typically flexible and variable. But in a certain situation, such as chemical treatment, biological functions are changed, and morphological changes also occur. This is the reason why studying cell surface fluctuations are crucial for the understanding of cell function about cell dynamics. Given the general nature of these physicochemical mechanisms, we expect that similar effects of PFA treatment around the elastic modulus and membrane fluctuations would also be expected although the specific magnitudes and responses conferred upon PFA treatment might vary on an absolute scale. We have Gadodiamide inhibitor confidence in that the SPM techniques could well serve as a encouraging tool for quantitative studies of both fixed cells and live cells in order to further explore this fascinating topic at the convergence Gadodiamide inhibitor of biology and nanotechnology. Methods Cell sample We used mouse fibroblast L929 cells (ATCC, USA) cultured in Dulbeccos altered eagle medium (DMEM; Invitrogen Life Technique, US) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, US) and 1% penicillin/streptomycin (Invitrogen Life Technique, USA) at 37?C in a humidified atmosphere containing 5% CO2. The cell samples with cell densities of 1 1??104/mL on a 35?mm diameter cell culture petri dish (NUNC, Denmark), were washed with phosphate buffered saline (PBS, Sigma-Aldrich, US) three times and then treated with different PFA solutions Gadodiamide inhibitor (and are the position of the pipette and the sample, respectively. The are the time-average position of cell surface and the deviation of the sample fluctuation. The non-fluctuation ion-current relation is approximately expressed as the following form : is the reference current when the pipette is usually far enough from your sample surface, and is a constant from your pipette geometry. It is here assumed that this cell fluctuation obeys the Gaussian distribution, and were decided experimentally to be is usually expressed as. is the Youngs moduls, is the Poissons ratio and is the indentation (depth). and alpha were set to be 0.5 and 35, respectively. The scan rate of the AFM cantilever and the maximum loading force were set to be 1C2?m/s and 3C8?nN, respectively. Cell viability assay To evaluate the viability of cells with PFA treatment, we used a LIVE/DEAD? Viability/Cytotoxicity Kit (L3224; Invitrogen life technique, USA). Briefly, the PFA-treated cells were immediately incubated using the live and lifeless stain fluorescence dye for 10?min. Then the final 2?M calcein AM and 4uM EtD-1 combination solution were added to the PFA-treated cell sample. A commercial fluorescence microscope (Nikon Corp., Japan) was used to obtain fluorescence images of cells where green and reddish colors represented live and lifeless cells, respectively. Authors contributions SOK and NJC designed the study; SOK conducted all experiments and performed data analysis; JK, TO and NJC assisted with data analysis; SOK, TO and NJC published the manuscript. All authors go through and approved the final manuscript. Acknowledgements The authors would like to thank the LIF Japan Society for the Promotion of Science, the National University or college of Singapore, and Nanyang Technological University or college promoting collaborative science project across Singapore and Japan. Competing interests The authors declare that.