In situ fixation and subsequent collection of cultured endothelial cells in a shear flow

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. Method name: In situ fixation and subsequent collection of cultured endothelial cells in a shear flow, Keywords: Endothelial cells, Endothelium, Adherent cells, In situ fixation, Cell dissociation, Mechanogenomics, Hi-C, Chromatin conformation capture