Influence of Fixation and Permeabilization on the Mass Density of Single Cells: A Surface Plasmon Resonance Imaging Study.

Ruoyu Cheng,Wei Wang,Yu-Wen Su,Feng Zhang,Xiang Wo,Meng Li

doi:10.3389/fchem.2019.00588

Abstract

Fixation and permeabilization of cells and tissues are essential processes in biological techniques like immunofluorescence and immunohistochemistry for cell biology studies. In typical procedures, the biological samples are treated by paraformaldehyde and Triton X-100 to achieve cellular fixation and permeabilization, respectively, prior to the incubation with specific antibodies. While it is well-known that the integrity of cell membrane has been broken during these processes, quantitative studies on the loss of cellular mass density and the enhancement of molecular accessibility at single cell level are still rare. In this study, we employed the surface plasmon resonance (SPR) imaging technique to monitor the mass density change of single cells during sequential fixation and permeabilization processes. We further utilize the osmotic responses of single cells to sugar molecules as an indicator to evaluate the integrity of cell membranes. It was found that, while fixation initially destructed the integrity of cell membranes and increased the permeability of intra- and extra-cellular molecules, it was permeabilization process that substantially induced significant loss in cellular mass density.

Highlights

Immunofluorescence is a powerful technique to visualize the distribution of specific biomolecules within biological samples such as cells and tissues (Joshi and Yu, 2017)
The fixation and permeabilization steps of cells and tissue samples, which could alter the permeability of cell membrane, are crucial procedures that could determine the successes of immunofluorescent or immunohistochemical assays
surface plasmon resonance (SPR) has the feature that both the resonant angle and the refractive index (RI) near the sensing surface are highly sensitive to the mass density of the surface layer in the medium-metal interface

Summary

Introduction

Immunofluorescence is a powerful technique to visualize the distribution of specific biomolecules within biological samples such as cells and tissues (Joshi and Yu, 2017). In typical cell-based immunofluorescent assays, adherent cells were incubated with fluorescent antibody to enable specific recognition and binding between the antibody and the target molecule in the cells. In order to facilitate the accessibility of antibody to the target molecules and to inhibit the inherent cellular activity, the samples were often fixed and permeabilized prior to the staining procedures. They were necessary when the target molecules were located within the cytoplasm. Among many types of reagents, paraformaldehyde (PFA) and Triton X-100 are probably the most widely used ones for fixation and permeabilization, respectively

Methods

Results

Conclusion