Glutaraldehyde fixation preserves the trend of elasticity alterations for endothelial cells exposed to TNF‐α

Abstract

Among the users of atomic force microscopy based techniques, there is an ongoing discussion, whether cell elasticity measurements performed on fixed cells could be used for determination of the relative elasticity changes of the native (unfixed) cells subjected to physiologically active external agents. In this article, we present a case, for which the legitimacy of cell fixation for elasticity measurements is justified. We provide an evidence that the alterations of cell elasticity triggered by tumor necrosis factor alpha (TNF-α) in EA.hy926 endothelial cells are preserved after glutaraldehyde (GA) fixation. The value of post-fixation elasticity parameter is a product of the elasticity parameter obtained for living cells and a constant value, dependent on the GA concentration. The modification of the initial value of elasticity parameter caused by remodeling of the cortical actin cytoskeleton is reflected in the elasticity measurements performed on fixed cells. Thus, such fixation procedure may be particularly helpful for experiments, where the influence of an external agent on the cell cortex should be assessed and AFM measurements of living cells are problematic or better statistics is needed.

Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy

“…PFA treatment affects the cross-linking of cell surface proteins, including F-actin filaments [5, 9, 17, 20, 21]. Taken together, we concluded that the stiffening of cells directly results in the cross-linking of proteins.…”

Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis

“…Despite ongoing controversy, there is increasing evidence suggesting that glutaraldehyde fixation is the preferred method for reliable data analysis (reproducible) of both cell elasticity and cell morphology [23,30–32]. …”

Nano-mechanical model of endothelial dysfunction for AFM-based diagnostics at the cellular level

“…The application of various 29 forms of physical stress, namely shear forces from the blood vessel 30 flow, periodic mechanical stimulation from blood pressure 31 changes, and contraction and expansion of the underlaying tissue, 32 to the cells trigger various mechanotransduction processes, 33 resulting in modifications in the cell biochemical activity and 34 structural rearrangements [1]. It is therefore apparent that 35 knowledge of the mechanical properties of the endothelium at 36 the cellular level is necessary for an accurate understanding of the 37 functionalities and mechanical response of the cells to both the 38 native and pathological environments.…”

“…[29] and by our group [30]. In 341 the work conducted by Tagosz-Korecka et al [30], the measurement 342 of the elasticity changes induced by TNF-a in living EA.hy926 343 endothelial cells were compared with the analogous data obtained 344 for cells fixed with glutaraldehyde (see panel A in Fig.…”

“…For a more detailed explanation of this 352 finding, please refer to Ref. [30]. incubation with TNF-a, and thus, this cytokine was chosen for the 373 study.…”