Single-cell lipidomics: protocol development for reliable cellular profiling using capillary sampling.

Single-cell lipidomics enables detailed analysis of the lipidomes of cells, but is challenged by small sample volumes, the risk of background interference and a lack of validation data. In this study, we explore the effect of different sampling variables on the lipid profiles of single pancreatic cancer cells, detected using liquid chromatography-mass spectrometry (LC-MS). We use automated and manual capillary sampling methods to isolate living single cells and evaluate different sampling media, capillary tips, aspiration volume, and temperature and humidity control. We demonstrate that automated and manual capillary sampling yield comparable lipid profiles when key parameters are controlled. Our findings highlight that appropriate blank correction, capillary tip type, and the control of aspiration volumes are all critical to preserving detection sensitivity. Conversely, choice of sampling medium does not affect lipidomics results. We also set out suggested best practices for these methodological variables, laying a foundation for robust, adaptable workflows in single-cell lipidomics for applications such as biomarker discovery and metabolic research.