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

## TL;DR

This paper provides a detailed protocol for using stable isotopes to study fat production in induced neural stem cells.

## Contribution

A new protocol for multi-omics analysis of iNSCs using isotopic tracing and mass spectrometry.

## Key findings

- A protocol for culturing and preparing iNSCs for multi-omics profiling.
- Steps for isotopic labeling and sequential extraction of metabolites, lipids, and proteins from one sample.
- Guidance for UHPLC-MS data acquisition and analysis.

## Abstract

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

•Instructions for culturing and preparing iNSCs for multi-omics profiling•Steps for stable isotopic labeling with [13C6]-glucose and [13C5,15N2]-glutamine•How to sequentially extract metabolites, lipids, and proteins from one sample•Guidance on UHPLC-MS data acquisition and analysis

Instructions for culturing and preparing iNSCs for multi-omics profiling

Steps for stable isotopic labeling with [13C6]-glucose and [13C5,15N2]-glutamine

How to sequentially extract metabolites, lipids, and proteins from one sample

Guidance on UHPLC-MS data acquisition and analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.