Endothelial-derived microparticles(MP) have the potential to become important diagnostic and therapeutic tools. Elevated levels of MP have been seen in a broad range of diseases. This study aimed to standardise the methods of detection and analysis of MP by optimising immuno-gold labelling and negative staining protocols and by utilising transmission electron microscopy (TEM) techniques to study size distribution and the presence of various surface markers such as annexin V, lactadherin, CD54 and CD105.
This in-vitro model was derived via the stimulation of the human cerebral microvascular endothelial cell line (hCMEC/D3) with tumour necrosis factor (TNF) to increase the number of MP being released. MP can be differentiated from other extracellular vesicles by their size, which ranges between 0.1-1μm. Surface markers were labelled with colloidal gold tags to identify individual markers within a purified MP population. The samples were then negatively stained using an optimised protocol to enhance contrast in resulting TEM studies.
The process of immuno-gold labelling and subsequent analysis is technically challenging and may be labour intensive. The benefits are that the MP can be imaged whole as opposed to requiring resin embedding and sectioning, which can negatively affect image interpretation. The chemical fixation methods used in this technique may affect the structural integrity of the samples, which needs to be taken into consideration.
A correlation between the number of surface markers and the size of MP has been established. Future studies into novel methods of preparing MP using cryo-preparation techniques such as cryo-sectioning could possibly enable improved structural preservation and immuno-gold labelling of markers located within MP.