A robust and efficient microvascular isolation method for multimodal characterization of the mouse brain vasculature

Studying disease related changes in the brain vasculature is pivotal due to its crucial role in supplying oxygen and nutrients, and removing waste. To study the vascular cells in detail, improved techniques for isolation of the vascular components are warranted. In this study, 22,515 cells were isolated using CD31 coupled magnetic beads and analyzed using single cell RNA sequencing. Their transcriptomes separated into 23 clusters corresponding to all known vascular and perivascular cell types. Western blot analysis using markers for these cell types confirmed their presence in the isolate and suggests that the protocol is suitable also for proteomic analysis. Finally, we adapted the protocol to accommodate primary culture. In conclusion, this method can successfully isolate murine brain microvasculature independent of cell sorting, alleviating the need for reporter mouse lines. The protocol is suitable for several testing modalities, including single-cell analyses, WB, and primary cell culture. Overall design: The samples are mouse brain vasculature, freshly isolated and digested into single cell suspensions. These were subjected to the 10x Genomics Gene expression v3.1 chemistry and sequenced on a NextSeq platform as per manufacturers' recommendations.