Quantifying Adipose Tissue Thermogenesis Using Highly Sensitive Isothermal Microcalorimetry

Isothermal microcalorimetry enables direct measurement of thermogenic heat production, but optimal conditions for its application to adipose tissue have not been systematically defined. Here, we establish experimental parameters for quantifying thermogenesis using the CalScreener, a high-throughput isothermal microcalorimetry platform, across adipocyte organoids, freshly isolated adipocytes, and intact adipose tissue explants. Heat output scaled with spheroid size within a defined range and increased linearly with spheroid number per well, improving measurement consistency. Freshly isolated adipocytes and intact adipose tissue exhibited robust and depot-specific thermogenic activity that was sensitive to physiological modulation. Notably, intact adipose tissue retained functional thermogenic capacity for at least 6 hours <i>ex vivo</i> when maintained in nutrient-containing medium, enabling batch analysis of large cohorts. In contrast, storage conditions designed to preserve mitochondrial integrity during cold handling suppressed basal heat production and did not maintain integrated thermogenic activity, despite preserved adrenergic responsiveness. Together, these results provide practical guidance for tissue handling, assay design, and interpretation of isothermal microcalorimetry-based measurements of adipose tissue thermogenesis.