Abstract
BackgroundCell 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, CPFA, is 4%. However, it is still not quantitatively clear how the conventional protocol of CPFA is optimized.MethodsHere, we investigated the mechanical properties of cell fixation as a function of CPFA by using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect of CPFA (0–10 wt%) on the morphological and mechanical properties of live and fixed mouse fibroblast cells.ResultsWe found that both Young’s modulus, E, and the fluctuation amplitude of apical cell membrane, am, were almost constant in a lower CPFA (<10−4%). Interestingly, in an intermediate CPFA between 10−1 and 4%, E dramatically increased whereas am abruptly decreased, indicating that entire cells begin to fix at CPFA = ca. 10−1%. Moreover, these quantities were unchanged in a higher CPFA (>4%), indicating that the cell fixation is stabilized at CPFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols.ConclusionsTaken 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.
Highlights
Cell fixation is an essential step to preserve cell samples for a wide range of biological assays involv‐ ing histochemical and cytochemical analysis
Understanding the detailed process of cell fixation in various states from living cells to completely fixed cells provides an opportunity to optimize cell fixation protocols and to gain useful knowledge about the living cell fixation process. To address this outstanding question, we investigated the mechanical properties of cell surface structures as a function of the concentration of PFA (CPFA) by using atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM)
These methods allow us to measure the elastic modulus and the surface fluctuation amplitude, respectively, of cells in both living and fixed states [13,14,15,16]. These measurement approaches can be applied to living cells to investigate cell mechanical changes in response to the PFA concentration, CPFA. We found that both cell stiffness and cell surface fluctuation underwent a transition around CPFA = 10−1–4%, and these quantities were unchanged at a higher CPFA (>4%), indicating that the cell fixation is stabilized at CPFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols
Summary
Cell fixation is an essential step to preserve cell samples for a wide range of biological assays involv‐ ing 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, CPFA, is 4%. Understanding how cells behave at material interfaces holds wide importance for key biological applications such as cell–material surface interactions [1], mechanobiology [2], and advanced cell analysis [3]. Among such applications, one of the most practical and widely methods used across the biological sciences is cell fixation, which is an essential process for histological analyses in clinical diagnosis. Understanding the detailed process of cell fixation in various states from living cells to completely fixed cells provides an opportunity to optimize cell fixation protocols and to gain useful knowledge about the living cell fixation process
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