Bio-maleimide-stained plasma microparticles can be purified in a native state and target human proximal tubular HK2 cells

Abstract

Plasma microparticles (MPs) are heterogeneously sized submicron extracellular vesicles that originate from the cell membrane as a result of cell activation or apoptosis. Circulating MPs express cell-specific molecules that reflect their cell of origin and they are increasingly investigated for their potential role in intercellular communication. The aim of the current study was to determine if size exclusion chromatography could be used to purify fluorescent-labeled MPs in sufficient concentrations to be used experimentally in cell binding assays. Bio-maleimide was used to stain plasma MPs in platelet free plasma before applying to size exclusion chromatography. Collected fractions were analyzed for protein content and MPs were enumerated by flow cytometry. Fractions were ultracentrifuged and MPs further confirmed by western blotting for the putative diabetic marker, cluster of differentiation (CD)36 and platelet-specific CD41 proteins. Fractions that contained MPs were incubated with HK2 cells to determine MP-cell binding. Bio-maleimide-stained MPs were detected across various fractions of size exclusion, and pellets of these fractions confirmed positivity for the MP markers, CD41 and CD36. The addition of the isolated MPs to HK2 renal tubular cells and analysis by epi-fluorescent imaging demonstrated that, in principle, the labeled MPs are able to bind to cells in vitro. Notably, only the first eluted MP fraction bound HK2 cells indicating a possible association between MP size and cell-targeting properties.