Abstract
In situ fixation of adherent cells is a necessary process for downstream assays. Current methods to dissociate adherent endothelial cells require the use of a cell scraper that may introduce variability in nuclear morphology. Also, a cell scraper is not an option for experiments using sealed flow chambers. HMEC-1 cells were sheared at 5 dyn/cm2 for 24 h and then fixed in situ, quenched, and dissociated at the same shear rate. Analysis revealed no statistically significant change in nuclear shape between the steps of fixation and dissociation. This method outlines an alternative for the dissociation of adherent sheared endothelial cells after being fixed in situ in a micro-scale channel without causing a change in the nuclear morphology.•This method can be used with any commercially available, or custom-made, flow chamber and flow system.•Allows for downstream experimentation with adherent cells fixed in situ, such as Hi-C analysis, without impacting nuclear morphology or chromatin organization.•Cells are cultured, fixed, and dissociated at the same shear rate. Using the same shear rate for each step yields results that are not influenced by variable forces.
Highlights
The linkage of nuclear morphology and cellular morphology suggests that crosslinking adherent cells while they are still adhered to the culturing surface is necessary to preserve global nuclear organization [7]
In this paper we present a method for fixing adherent sheared endothelial cells in situ and collecting these cells under the same shear forces used during the experiment for Hi-C analysis
Individual Regions of Interest (ROI) data were reviewed in comparison to the original images and was excluded from the data set if it met the following exclusion criteria: (1) the nucleus had non-distinct edges as classified by a noticeable blurring around the cell that would indicate the nucleus was not in focus; (2) failure of the processing protocol to appropriately watershed at the junction of two or more nuclei; and, (3) an over-saturation of the DAPI fluorescence signal that could affect the detection of the nuclear shape
Summary
The cellular microenvironment plays a substantial role in cellular function and genome regulation [1]. To our knowledge, there is no effective way to study changes in nuclear shape or chromatin organization in adherent endothelial cells that are fixed in situ. The purpose of this protocol is to develop an effective method to collect adherent endothelial cells to be used for Hi-C analysis. In this paper we present a method for fixing adherent sheared endothelial cells in situ and collecting these cells under the same shear forces used during the experiment for Hi-C analysis This method allows adherent cells to be collected while maintaining both the morphology of the cells and nuclei developed during exposure to fluid shear stress
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