An improved method for the precise unravelment of non-shivering brown fat thermokinetics.

Since the discovery of functional brown adipose tissue (BAT) in adult humans, research on BAT gained a new popularity to combat obesity and related comorbidities. To date, however, methods to quantify BAT thermogenesis are often either highly invasive, require advanced equipment, are time consuming or of limited sensitivity. Here we present a simple yet highly effective and minimally invasive protocol for the Precise Unravelment of Non-shivering brown fat thermoKinetics (PUNK) in mice using infrared thermography in combination with Vaseline to brush up the fur between the shoulder blades. We also use physiological and molecular readouts including indirect calorimetry, qPCR and Western Blots to test our protocol. Our study demonstrates that Vaseline before thermography vastly advances the reproducibility and quality of BAT infrared pictures or videos, as it exposes the skin above the BAT for a direct line of sight for the infrared camera and thereby circumvents the well-known problems associated with shaving and anaesthesia. We subsequently validate that this approach does not affect physiological and molecular BAT function, but instead leads to more robust and less variable results when comparing for instance norepinephrine stimulation tests or knockout animals. Taken together, the PUNK protocol for BAT thermography quickly and effectively improves scientific outcomes of this method, and can be easily added to existing paradigms. Consequently, it safes money, time and experimental animals, thereby putting the 3R's principles of animal welfare into practice.