Protocol for stable isotopic tracing to assess cellular lipogenic activity in induced neural stem cells

Here, we present a protocol to assess the lipogenic phenotype of induced neural stem cells (iNSCs) using stable isotopic tracing. We describe steps for the culture and preparation of iNSCs, labeling with [¹³C₆]-glucose and [¹³C₅, ¹⁵N₂]-glutamine, and the subsequent extraction of metabolites, lipids, and proteins from the same sample. This protocol supports single-specimen, mass spectrometry-based multi-omics workflows and is applicable to steady-state analyses, stable isotope tracing, and characterization of protein post-translational modifications. For complete details on the use and execution of this protocol, please refer to Ionescu et al.¹.