Rectal probe

Mice received 1–2 intraperitoneal (i.p.) injections of the indicated amounts of acrylamide or glycidamide in DPBS or vehicle at a volume of 10 μL/g mouse body weight. Core body temperature was measured using a rectal probe (Physitemp Instruments). For chronic oral exposure, mice first received 50 ppm acrylamide in drinking water ad libitum for two days to acclimate mice to acrylamide taste, thus avoiding dehydration secondary to water aversion. Following this acclimation period, mice were exposed to the indicated dose of acrylamide throughout the experimental period.

Ten‐ to 11‐week‐old C57Bl/6J mice were injected with 1 × 10⁶ cancer cells or 1× PBS as vehicle control into the musculus gastrocnemius of the right hind leg. At study endpoints blood was drawn via the retro‐orbital plexus, mice were sacrificed by cervical disocation, and tissues excised and snap‐frozen in liquid nitrogen. In vivo body‐ and tumour composition was assessed using NMR and MRI, respectively (Supplemental Methods). Body temperature was assessed using a rectal probe (Physitemp, NJ, USA). Systemic metabolism was analysed using a laboratory animal monitoring system as described (Supplemental Methods).

For cold exposure, mice were placed individually in a room with the temperature set at 4°C for 7 days. The animals had free access to food and water during this period. Their core body temperature was measured using a rectal probe (Physitemp Instruments Inc., USA). CL316,243 (Sigma-Aldrich, USA) was injected interperitoneally at a dose of 1 mg/(kg·day⁻¹), and the injection protocol was described in the results part for details.

C57Bl/6 mice were housed under standard L:D-conditions (L:D cycle of 12 h:12 h) in the animal facilities of the University of Groningen, The Netherlands. Prior to experiments, animals were fed ad libitum using standard animal lab chow. Torpor was induced pharmacologically by injecting 7.5 mmol/kg of 5′-AMP (Sigma Aldrich) in 0.9% saline (pH 7.2–7.5) intra-peritoneally. To record body temperature during experiments, we measured the body temperature using a rectal probe (Physitemp Instruments). Mice were euthanized at different times after injection of 5′-AMP or saline. The minimum body temperature during torpor was reached at 4–5 hours following 5′-AMP injection and full arousal with normalization of body temperature occurred by 10 hours after 5′-AMP administration. At euthanization, animals were anesthetized using 3% isoflurane/oxygen and up to ∼800 μl blood was drawn immediately by abdominal aortic puncture into 3.2% sodium citrate and small EDTA-coated tubes. Automated hematological analysis was performed within 5 hours using a Sysmex XE-2100 [29] (link). The platelets were discriminated from other cells by Forward and Sideward Scatter characteristics. Mature and immature platelets were separated on the basis of Side Scatter, by virtue of the increased amount of granular (i.e. scattering) organelles in immature platelets.

All procedures were performed in accordance with the guidelines stipulated in a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas at Dallas. Mice were anesthetized with 1-2% isoflurane (Vedco, St. Joseph, MO) and restrained in the prone position. After confirming the depth of anesthesia by toe pinch, the animals were catheterized via either the left or right lateral tail vein using a winged butterfly infusion set (Terumo Corporation, Tokyo, Japan). Whole body temperature of the mice was maintained at 37 °C using a closed loop temperature control system comprising a heat lamp and a rectal probe (Physitemp Instruments, Clifton, NJ). Following sedation and catheterization, mice were transferred from the prep station to a custom 3D printed imaging stage, outfitted with a circulating water bath (T/Pump, Stryker, Kalamazoo, MI), for further treatment.