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      저자 : Okajima Takaharu (검색결과 1 건)
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      • Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy

        Kim Seong-Oh,Kim Joonhui,Okajima Takaharu,Cho Nam-Joon 나노기술연구협의회 2017 Nano Convergence Vol.4 No.5

        Background Cell fixation is an essential step to preserve cell samples for a wide range of biological assays involving histochemical and cytochemical analysis. Paraformaldehyde (PFA) has been widely used as a cross-linking fixation agent. It has been empirically recognized in a gold standard protocol that the PFA concentration for cell fixation, C PFA, is 4%. However, it is still not quantitatively clear how the conventional protocol of C PFA is optimized. Methods Here, we investigated the mechanical properties of cell fixation as a function of C PFA by using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect of C PFA (0–10 wt%) on the morphological and mechanical properties of live and fixed mouse fibroblast cells. Results We found that both Young’s modulus, E, and the fluctuation amplitude of apical cell membrane, a m, were almost constant in a lower C PFA (<10−4%). Interestingly, in an intermediate C PFA between 10−1 and 4%, E dramatically increased whereas a m abruptly decreased, indicating that entire cells begin to fix at C PFA = ca. 10−1%. Moreover, these quantities were unchanged in a higher C PFA (>4%), indicating that the cell fixation is stabilized at C PFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols. Conclusions Taken together, these findings offer a deeper understanding of how varying PFA concentrations influence the mechanical properties of cells and suggest new avenues for establishing refined cell fixation protocols. Background Cell fixation is an essential step to preserve cell samples for a wide range of biological assays involving histochemical and cytochemical analysis. Paraformaldehyde (PFA) has been widely used as a cross-linking fixation agent. It has been empirically recognized in a gold standard protocol that the PFA concentration for cell fixation, C PFA, is 4%. However, it is still not quantitatively clear how the conventional protocol of C PFA is optimized. Methods Here, we investigated the mechanical properties of cell fixation as a function of C PFA by using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect of C PFA (0–10 wt%) on the morphological and mechanical properties of live and fixed mouse fibroblast cells. Results We found that both Young’s modulus, E, and the fluctuation amplitude of apical cell membrane, a m, were almost constant in a lower C PFA (<10−4%). Interestingly, in an intermediate C PFA between 10−1 and 4%, E dramatically increased whereas a m abruptly decreased, indicating that entire cells begin to fix at C PFA = ca. 10−1%. Moreover, these quantities were unchanged in a higher C PFA (>4%), indicating that the cell fixation is stabilized at C PFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols. Conclusions Taken together, these findings offer a deeper understanding of how varying PFA concentrations influence the mechanical properties of cells and suggest new avenues for establishing refined cell fixation protocols.

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