Uplanapo n a 1

Live-cell epi-fluorescence imaging (Figures 1A, 1B, 2D, and S1–S5) was performed on an IX70 inverted microscope (Olympus, Tokyo, Japan) equipped with an objective lens (60×, UplanApo N.A. 1.40; Olympus). The temperature of the culture medium was maintained at 37°C by controlling with a stage and a microscope objective lens heater with a controller (MI-IBC; Olympus). A cooled CCD camera (ORCA-ER; Hamamatsu Photonics, Hamamatsu, Japan) was used for the acquisition of cell images. The fluorescence images of EGFP and Alexa488 were taken using a sapphire laser (488-nm Model 488-30 CDRH; Coherent), a dichroic mirror (DM505; Olympus), and an emission filter (BA515-550; Chroma Technology, Yokohama Japan). The fluorescence images of Cy3 were taken with a green solid-state laser (532 nm Compass 315M-100; Coherent, Santa Clara, CA), a dichroic mirror (Q565LP; Olympus), and an emission filter (HQ610/75M; Chroma Technology). The fluorescence images of Cy5 and RFP were taken using a red He–Ne laser (633-nm GLS5360; Showa Optronics), a dichroic mirror (660LP; Olympus), and an emission filter (700/75M; Chroma Technology). Fluorescence images were quantitatively analyzed using AQUA-Lite ver. 10 (Hamamatsu Photonics) at various intervals after microinjection.

An intracellular thermogenesis assay was performed using two different thermoprobes. BAs (day 6) were washed with PBS and 5% glucose solution and then incubated with 0.04 wt/vol% cellular thermoprobe (FDV-0004; Funakoshi) for 10 min. The fluorescence intensity of single cells was observed before and after stimulation with 0.5 μM CL316,243 (Sigma-Aldrich) by confocal laser microscopy (TSC-SP8; Leica). A hydrophilic fluorescent nanogel thermometer (Gota et al, 2009 (link)) was microinjected into the cytosol of BAs. The fluorescence intensity of single cells was observed before and after stimulation with 10 μM CCCP (Sigma-Aldrich) under an IX70 inverted microscope (Olympus) equipped with an objective lens (60×, UplanApo N.A. 1.40; Olympus). A cooled charge-coupled device camera (ORCA-ER) was used to acquire cell images.