Protocol for measuring lipid membrane fluidity in human iPSC-derived neural cells

Here, we present a protocol for describing the differentiation of human cortical neurons from induced pluripotent stem cells (iPSCs) and the subsequent analysis of lipid membrane fluidity using the LipiORDER fluorescent probe. We describe steps for embryoid body formation, neuronal induction with adherent culture maturation, and live-cell membrane fluidity measurement. This approach allows for investigation of the effects of lipid membrane fluidity on neuronal function and pathophysiology. For complete details of the use and execution of this protocol, please refer to Morita et al. 1 • Instructions for differentiating human cortical neurons from iPSCs • Guidance on live-cell membrane fluidity measurement using LipiORDER • Steps to quantify membrane fluidity using ImageJ-based processing Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we present a protocol for describing the differentiation of human cortical neurons from induced pluripotent stem cells (iPSCs) and the subsequent analysis of lipid membrane fluidity using the LipiORDER fluorescent probe. We describe steps for embryoid body formation, neuronal induction with adherent culture maturation, and live-cell membrane fluidity measurement. This approach allows for investigation of the effects of lipid membrane fluidity on neuronal function and pathophysiology.