Protocol for generation of immortalized retinal, choroid, and brain endothelial cell lines.

The integrity and functionality of endothelial cells (ECs) play a crucial role in the homeostasis of different tissues; however, ECs show tissue-specific morphology and gene expression profile. Here, we present a protocol for the production of stable EC lines from mouse retinal, choroidal, and brain tissue. We outline the procedures for preparing the three specified tissues and generating single-cell suspensions through enzymatic digestion. Subsequently, we describe the methods used to selectively immortalize ECs and maintain their cultures for downstream experiments. For complete details on the use and execution of this protocol, please refer to Heiden et al.1.