Protocol for measuring mitochondrial respiration in mature human adipocytes using the Seahorse XF analyzer

Primary mature human adipocytes are an important tool bridging basic research and clinical medicine by reflecting human biological diversity. Here, we present a protocol for measuring the oxygen consumption rate of mature adipocytes. We describe the steps for isolation of adipocytes from adipose tissue samples, Matrigel embedding, and Seahorse analysis, as well as normalization and data analysis. This protocol enables the investigation of the metabolic function of primary mature human adipocytes in response to drugs or genetic modifications. • Steps for the isolation and handling of viable primary human mature adipocytes • Instructions for embedding of floating adipocytes in Matrigel for Seahorse analysis • Guidance on Seahorse XF setup for OCR and ECAR measurements of mature adipocytes • Procedure for normalizing OCR measurements tailored for primary mature adipocytes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Primary mature human adipocytes are an important tool bridging basic research and clinical medicine by reflecting human biological diversity. Here, we present a protocol for measuring the oxygen consumption rate of mature adipocytes. We describe the steps for isolation of adipocytes from adipose tissue samples, Matrigel embedding, and Seahorse analysis, as well as normalization and data analysis. This protocol enables the investigation of the metabolic function of primary mature human adipocytes in response to drugs or genetic modifications.