Difference in intracellular temperature rise between matured and precursor brown adipocytes in response to uncoupler and β-adrenergic agonist stimuli

Brown adipocytes function to maintain body temperature by heat production. However, direct measurement of heat production at a single cell level remains difficult. Here we developed a method to measure the temperature within primary cultured brown adipocytes using a cationic fluorescent polymeric thermometer. Placement of the thermometer within a matured brown adipocyte and a precursor cell enabled the detection of heat production following uncoupler treatment. The increase in the intracellular temperature due to stimulation with a mitochondrial uncoupler was higher in matured brown adipocytes than in precursor cells. Stimulation with a β-adrenergic receptor (β-AR) agonist, norepinephrine, raised the intracellular temperature of matured brown adipocytes to a level comparable to that observed after stimulation with a β3-AR-specific agonist, CL316.243. In contrast, neither β-AR agonist induced an intracellular temperature increase in precursor cells. Further, pretreatment of brown adipocytes with a β3-AR antagonist inhibited the norepinephrine-stimulated elevation of temperature. These results demonstrate that our novel method successfully determined the difference in intracellular temperature increase between matured brown adipocytes and precursor cells in response to stimulation by an uncoupler and β-AR agonists.


Results
Characteristics of primary cultured BACs. Differentiated BACs showed multilocular morphology, whereas primary undifferentiated pre-BACs from stromal vascular cells showed a fibroblast-like morphology (Fig. 2a). Almost all cells in the differentiation culture had accumulated several lipid droplets, indicating that the differentiation had occurred with high efficiency. Browning of primary adipocytes from stromal vascular cells of interscapular BAT can be evaluated by measuring the mRNA expression of UCP1 and other gene markers of matured BACs by qRT-PCR. We selected PGC-1α as a BAC-specific gene marker, which induces mitochondrial biogenesis and UCP1 expression, resulting in thermogenesis 40 . We also measured the mRNA expression of FABP4 and PPARγ, which are major adipocyte markers, and CPT-1β and PPARα, which are known markers of BACs. As shown in Fig. 2b, the expressions of UCP1 and PGC-1α mRNA were much higher in matured BACs than in pre-BACs. In addition, the mRNA expression levels of CPT-1β, FABP4, PPARα, and PPARγ were at least twice as high in BACs as in pre-BACs. To confirm that the protein level of UCP1 had increased in BACs, we performed western blot analysis and confirmed that UCP1 protein expression was strongly induced by differentiation in primary culture (Figs. 2c and S1a). In addition, the stimulation of BACs by 100 nM NE induced intense expression of UCP1 gene and protein (Figs. 2d, S1b and 2e), which is an essential functional characteristic of BACs 41 .
We next introduced the R-CFPT thermometer into BACs by referring to the previously established procedure for MOLT-4 (human acute lymphoblastic leukemia) and HEK293T (human embryonic kidney) cells 35 . We treated cells with 0.05 w/v% of R-CFPT in 5% glucose solution at 25 °C for 10 min. Representative differential interference At low temperature, the thermo-responsive polyNNPAM sequence in R-CFPT (green line) assumes an extended structure such that the water-sensitive DBThD-AA unit (gray star) can be quenched by neighboring water molecules. At higher temperature, the polyNNPAM sequence shrinks because of hydrophobic interactions among the NNPAM units (green line), resulting in the release of water molecules and strong fluorescence from the DBThD-AA unit (yellow star). The fluorescence intensity of the second fluorescent unit, BODIPY-AA (red star), is relatively insensitive to temperature-dependent changes in the polyNNPAM structure. The cationic APTMA unit (blue symbol) promotes incorporation of R-CFPT into the cell and enriches its hydrophilicity to prevent interpolymeric aggregation within a cell. contrast (DIC) and confocal fluorescence images of R-CFPT, Hoechst33342 (nucleus), NileRed (lipid), and MitoTrackerDeepRed (mitochondria) of BACs after treatment with R-CFPT are shown in Fig. 3a. As reported for HEK293T and MOLT-4 cells 35 , R-CFPT showed spontaneous uptake into the BACs. The R-CFPT-stained area did not overlap with the NileRed-stained lipid droplets or the MitoTrackerDeepRed-stained mitochondria, indicating that R-CFPT had diffused throughout the cytosol and the nucleus.
Next, to optimize the R-CFPT incorporation efficiency and cytotoxicity, BACs were incubated with various concentrations of R-CFPT (0.01, 0.03, 0.04 or 0.05 w/v%) in 5 w/v% glucose at 25 °C for 10 min, and the incorporation efficiency of R-CFPT was calculated from confocal fluorescence images taken at the various R-CFPT concentrations. As shown in Table 1, the incorporation efficiency increased as the relative proportion of R-CFPT Figure 2. Physiological properties of primary cultured brown adipocytes (BACs) in this study. (a) Representative differential interference contrast (DIC) images of pre-BACs and matured BACs. Bar, 50 µm. (b) UCP1, PGC-1α, CPT-1β, FABP4, PPARγ, and PPARα mRNA expression in primary culture of pre-BACs and matured BACs measured by RT-PCR (n = 3, mean ± SD). Data were normalized to the corresponding 18S rRNA values. (c) Western blot analysis of UCP1 protein expression in pre-BACs and BACs (20 μg protein from cell lysate). The exposure times to detect α-tubulin and UCP1 bands were 8 s and 60 s, respectively. (d) UCP1 protein expression in BACs (10 μg protein) in response to NE stimulation. The exposure times to detect α-tubulin and UCP1 bands were 15 s and 240 s, respectively. (e) Expression of UCP1 mRNA in BACs in response to NE stimulation as measured by RT-PCR (n = 3, mean ± SD). Data were normalized to the corresponding 18S rRNA values.   rose from 0.01 to 0.05 w/v%. Cytotoxicity was assessed by propidium iodide (PI) assay, which is generally used to estimate damage to the plasma membrane and serves as a marker of cell death 42 . Cytotoxicity was determined in cells cultured for 30 min after introduction of R-CFPT. As shown in Table 1, there was no significant difference in cell viability between non-R-CFPT treated cells and cells treated with R-CFPT at a concentration of 0.04 w/v% or lower. At 0.05 w/v% R-CFPT, some cells began to die, as evidenced by plasma membrane rupture (Table 1). Based on these results, we chose 0.04 w/v% R-CFPT for subsequent experiments to measure intracellular temperature, because this concentration showed sufficient cellular incorporation efficiency and almost no cytotoxicity.
In the case of pre-BACs, treatment at 0.05 w/v% of R-CFPT achieved probe uptake in nearly all of the cells (95.0 ± 4.7%) (Fig. 3b). We did not observe any dead pre-BACs in the PI assay under this condition. Due to the lack of cytotoxicity, the high incorporation efficiency and sufficient fluorescence intensity of intracellular R-CFPT under fluorescence microscopy observation at this high concentration of R-CFPT (0.05 w/v%), further optimization was not performed. Intracellular temperature change induced by uncoupler stimulation in BACs. We next used the R-CFPT probe with confocal microscopy observation to measure the intracellular temperature change in BACs. The ratio of the two fluorescence intensities of R-CFPT in a BAC lysate displayed a temperature-dependent increase (Fig. 4a). The temperature resolution, meaning the statistical minimum temperature difference that can be significantly discriminated, was 0.06 to 0.56 °C within the range 32 to 38 °C (Fig. 4a) when measured under a confocal microscope (see Methods). These results indicate that measured temperature changes can be discussed from the viewpoint of biological phenomena only if the temperature changes are larger than the temperature resolution. To confirm the functionality of R-CFPT within a BAC, we measured the heat production induced by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) 43 , which is known to accelerate thermogenesis in mitochondria 18 . Representative intracellular temperature imaging after FCCP treatment calculated from the ratio of two simultaneously acquired fluorescence images derived from DBThD-AA (560-610 nm) and BODIPY-AA (500-520 nm) is shown in pseudocolor in Figs. 4b and S2 in Supplementary Information. Whereas the DMSO control did not induce changes in the fluorescence ratio, FCCP increased the temperature throughout cells, as shown by the higher fluorescence ratio. Furthermore, the intracellular temperature increase induced by 30 μM FCCP was higher than that induced by 10 μM FCCP in matured BACs. The average cellular temperature changes calculated from the time-lapse images of R-CFPT fluorescence in the BACs indicated that heat was generated immediately after stimulation by the uncoupler FCCP (Fig. 4c). As shown in Fig. 4d, 10 μM FCCP also stimulated heat production in pre-BACs, whereas 30 μM FCCP caused cell death, as indicated by obvious cell morphological changes (Fig. S3), such that temperature data were not available. As shown in Fig. 4c and d, the maximum temperature elevation caused by 10 μM FCCP was 1.60 ± 0.11 °C for BACs (at 22 min, n = 9 cells, mean ± SE) and 1.20 ± 0.11 °C for pre-BACs (at 31 min, n = 16 cells, mean ± SE). A significant difference in the maximum temperature increase between BACs and pre-BACs was confirmed by t-test (p = 0.0268). Furthermore, when the temperature of the medium was maintained at 30 °C, the intracellular temperature of pre-BACs and BACs was significantly different at 32.3 ± 0.2 °C (mean ± SE, n = 22) and 34.4 ± 0.2 °C (n = 50), as confirmed by Student's t-test (p < 0.001). In contrast, the temperature of the culture medium during the experiments was monitored by R-CFPT and a thermocouple, and no changes in temperature were detected (Fig. S4). This indicates that the temperature increase in BACs was due to endogenous thermogenesis and not to global heating of the culture medium. Intracellular temperature change induced by β3-AR agonist stimulation in BACs. We next investigated whether stimulation with β-AR agonists would induce an intracellular temperature change in BACs. Representative intracellular temperature images after application of the β-AR agonists NE and CL316.243 are shown in pseudocolor in Figs 5a and S2. The results demonstrated that these β-AR agonists induced intracellular thermogenesis. As shown in Fig. 5b, the mean temperature increase at 31 min after NE and CL316.243 application was 1.25 ± 0.25 °C (mean ± S.E.) and 1.39 ± 0.38 °C, respectively. No significant difference in temperature change was observed between the two treatments. In contrast, no temperature change was observed in pre-BACs after β-AR agonist treatment (Fig. 5c), suggesting that intracellular thermogenesis induced by β-AR activation is specific to matured BACs.
To confirm the induction of thermogenesis by β3-AR activation, we evaluated the intracellular temperature of BACs pretreated with a specific β3-AR antagonist. Lipolysis assays indicated that SR59230A (10 μM) adequately suppressed NE-evoked lipolysis after 4 h of treatment, but not after 2 h of treatment (Fig. S5), which is consistent with the results of Tonello et al. 44 . The intracellular temperature of SR59230A-pretreated BACs remained almost constant despite NE stimulation (Fig. 5d). Statistical analysis also indicated that the cellular temperature change observed at 31 min after NE and CL316.243 treatment was significantly different from that after control treatment. In contrast, no significant differences in intracellular temperature change were observed between NE stimulation after pretreatment with SR59230A and the control (Fig. 5e). Together, these results strongly indicate that the thermogenesis induced by NE in BACs occurs through β3-AR activation. Finally, we evaluated whether there was a correlation between the cell temperature before stimulation and the degree of intracellular temperature change due to the stimulus (Fig. S6). No clear correlation was observed with any stimulus, including FCCP, NE and CL 316.243.

Discussion
In this study, we have optimized a method to measure the intracellular temperature in primary cultured BACs by a CFPT for ratiometric sensing. The phenotype of primary cultured BACs is highly dependent on the culture conditions and origins of the precursor cells. For example, under non-optimized conditions, no UCP1 expression is detected even after NE stimulation 45 . The primary cultured BACs used in this study showed significant UCP1 protein expression (Fig. 2c), BAC-specific gene expression (Fig. 2b), and increased expression of UCP1 after NE stimulation (Figs. 2d and e). Optimization of the conditions for application of R-CFPT to BACs demonstrated an incorporation of R-CFPT into 36.2% of cells with low cytotoxicity. In previous studies, a non-ratiometric and cationic thermometer, termed FPT, was incorporated into approximately 50% of NIH3T3 cells and COS7 cells 20 , while R-CFPT was incorporated into almost all MOLT-4 and HEK293T cells 35 . Thus, introducing the polymeric thermometer into primary cultured BACs seems to be more challenging than its introduction into other cell lines.
The temperature resolution of R-CFPT evaluated in a BAC extract under microscopy-that is, 0.06 to 0.56 °C in the temperature range 32 to 38 °C (Fig. 4a)-seems to be sufficient for detecting the production of heat within BACs. The increases in cellular temperature were almost 1 °C or more during stimulation with FCCP or NE (Figs. 4c and 5b). The high temperature resolution of our method revealed differences in the intracellular temperature at steady state between matured BACs and pre-BACs.
To test the fluorescent polymeric thermometer, we examined whether uncoupling the mitochondrial membrane potential would lead to an increase in intracellular temperature. Plotting the time course of average cellular temperature changes calculated from the time-lapse images of R-CFPT in BACs indicated that heat generation was induced immediately after stimulation with the uncoupler FCCP (Fig. 4c). This result is comparable to that of a previous report in which an FCCP stimulus initiated a significant drop in mitochondrial inner membrane potential within 5 min of application, with a continued decrease in the potential for 30 min 46 . Moreover, in a recent study, treatment with the uncoupler CCCP, an analog of FCCP, was shown to increase neuronal cell temperature within 1 min of application 25 . Thus, our findings show that an uncoupler induces rapid heat generation in BACs comparable to its effect in other cell types.
Previous reports using several intracellular thermometers based on different detection systems have indicated that uncoupler treatment increases the intracellular temperature 18,21,25,27,28,34,47 . In this study, we demonstrated a difference between the intracellular temperature of matured BACs and that of pre-BACs. Furthermore, the increase in intracellular temperature at 31 min after application of 30 μM FCCP to BACs (mean 3.46 °C) was much higher than that reported previously for any other cell type or for pre-BACs in this study. This large temperature increase may be caused by the numerous mitochondria in matured BACs, as an uncoupler induces the release of heat from the mitochondria due to inhibition of ATP synthesis 48 . The concentration of mitochondrial protein is 20-to 30-fold higher in matured BACs than in pre-BACs 49 . One reason for the weak correlation between the mitochondrial mass and the increase in intracellular temperature may be the different expression of UCP1 protein in mitochondria in the two cell types; the mitochondria of the BACs used in this study dissipate heat modestly even in the unstimulated state due to the expressed UCP1 protein. In fact, the baseline cellular temperature of matured BACs was approximately 2 °C higher than that of pre-BACs. The influence of UCP1 on the intracellular temperature will be clarified in future studies using UCP1 knock-out or knock-down cells.
Next, we focused on the cellular temperature change induced by β3-AR stimulation. CL316.243, a selective β3-AR agonist, increased the intracellular temperature by the same degree as NE stimulation (Fig. 5b). Furthermore, although NE activates β1-, β3-, and α1-ARs, which are all expressed on the surface of BACs 50 , the β3-AR antagonist SR59230A inhibited the heat production stimulated by NE. Thus, our intracellular thermometry experiments demonstrated that β3-AR activation induces thermogenesis, which is consistent with the results of previous reports 51,52 . Further, the temperature increase induced by the β3-AR agonists occurred only in matured BACs, not in pre-BACs, demonstrating that thermogenesis in response to β3-AR agonist stimulation is specific to matured BACs.
Other recent studies have used various methods to measure changes in BAC temperature due to β3-AR activation 22,27,34,53 . Sato et al. used a temperature-responsive microcantilever to detect an increase of approximately 0.2 °C in cellular temperature during stimulation with 1 μM NE (>60 min) 53 , whereas the temperature increase in our study was 1.2 °C at 30 min after NE stimulation (Fig. 5b). As discussed by Sato et al. 53 , the temperature increase in their study was relatively small because their experiments were conducted at low temperature (25 ± 1 °C). In addition, there are likely other differences between our study and theirs, such as the physiological condition of the BACs derived from preparation. Consumption of O 2 reaches a peak within 5 min of NE application to BACs 54 . However, the heat generation at 30 min was also relatively low in the experiment using a microcantilever 53 .
Kriszt et al. reported the microscopic visualization of thermogenesis in human brown adipocytes using a small organic fluorescent molecule accumulating in the ER 34 . The temperature of the ER in matured brown adipocytes was also elevated in response to β3-AR activation while precursor cells showed no changes in ER temperature. Hattori et al. reported that the intracellular temperature of BACs was elevated by approximately 1.3 °C in response to CL316.243 stimulation using hydrophilic fluorescent nanogel thermometers, which are localized in the cytoplasm 22 . This elevation is comparable to the intracellular temperature increase we observed using R-CFPT localized in the nucleus and the cytoplasm. These results suggest that the intracellular temperature change in response to CL316.243 occurred throughout the BACs. As Kiyonaka et al. 27 reported, the temperature within BAC mitochondria is also elevated by stimulation with 10 μM NE. Although there is little description of the quantitative temperature rise in this report, the calibration curve suggests a mitochondrial temperature increase of a few degrees. As observed by transmission electron microscopy, the mitochondrial volume density of matured BACs is approximately 30% per cell volume 55 . In addition, in the present study, the intracellular temperature was calculated from the average of ratio intensity per entire single cell (see Methods). The NE-induced increase in intracellular temperature measured in this study is comparable to that measured by a fluorescent protein sensor 27 . The temperature rise of BACs by several degrees due to the β3-AR stimulation could be confirmed despite methodological differences (microcantilever 53 , small organic molecule 34 , polymer nanogel 22 and a fluorescent protein 27 ), suggesting that the intracellular temperature elevation is not an artifact.
Regarding the intracellular temperature distribution, our results showed that the temperature of nuclei (Figs. 4b and 5a) was higher than that of cytoplasm, which is consistent with the findings of previous reports in COS7 and HeLa cells with other polymeric thermometers and a thermometer consisting of two fluorescent proteins 18,20,28 . Previous reports showed that the temperature in the vicinity of mitochondria in COS7 cells and the temperature of the ER in brown adipocytes also increase for 30 min or longer after stimulation by FCCP and forskolin 18,34 . These findings are consistent with observations in the present study (Figs. 4b and 5a). We speculate that a constant thermodynamic steady state has been maintained due to continuation of heat production from mitochondria.
Finally, we discuss the validation of our intracellular thermometry using R-CFPT. Our results showed that the intracellular temperature of BACs was elevated by ~3 °C by chemical stimulation (FCCP and NE). The temperature measurements outside the cells indicated that the intracellular temperature increase was due to endogenous thermogenesis and not from an external heat source (Fig. S4 in Supplementary Information). Baffou et al. previously stated that a single biological cell does not have sufficient energy for a temperature elevation of 1 °C based on the conventional law (Equation 1): where P is the heat power, κ is the thermal conductivity of cells, and L is the diameter of the heat source 56 . The reason for the discrepancy between experimental data and the theoretical value derived from Eq. 1 has often been discussed in the field of intracellular thermometry. In fact, the temperature increase in a single live cell has been measured in various kinds of cell lines using thermometers with different functional mechanisms: a few degrees in mitochondria in BACs by GFP 27 ; 0.2 °C in BACs by a microcantilever 53 ; 0.6 °C in U251 cells by a thermocouple 57 ; 1.8 °C in NIH/3T3 cells by a quantum dot 24 ; and 1.7 °C in ER in HeLa cells by an organic fluorescent molecule 33 . The commonality in the intracellular temperature increase strongly suggests that the observed temperature increases in cells are not artifacts. Furthermore, Baffou et al. stated that cellular temperature changes measured by fluorescence-based thermometry reflect factors other than the temperature, such as pH and ion strength. However, no alteration in the cell size of brown adipose cells that could cause a rapid change of intracellular ion-strength was observed during both uncoupler and β3-AR stimulation (Figs. 4b and 5a). The previous report also showed that the stimulation using NE changed the intracellular pH in BACs by only 0.2 58 . Considering the independence of the fluorescence properties of R-CFPT from pH and ionic strength 35 , this suggests that the temperature change in BACs observed in this study does not reflect changes in intracellular pH and ionic strength.
Careful evaluation of each term in Eq. 1 might satisfy both experimental and theoretical researchers. Baffou et al. used 0.1 nW for the heat source of a single cell, but it has been reported that BACs produce much more heat (~1.6 nW) per cell 59,60 . In addition, the thermal conductivity of a biological cell is estimated to be larger than that of water, which was used in Eq. 1 in the original discussion. Further experimental data on cellular thermodynamics will be helpful in establishing that a single cell can substantially raise its temperature by endogenous thermogenesis.
In summary, here we have developed a method to measure temperature within primary cultured BACs using a CFPT. The significant difference in responsiveness to the stimulation of an uncoupler and β-AR agonists between undifferentiated cells and mature cells was observed by analyzing average temperature changes of whole cells.
Stimulation with a β3-AR agonist increased the temperature of matured BACs by approximately 1 °C, but did not affect immature pre-BACs. Furthermore, pretreating mature BACs with a β3-AR antagonist completely inhibited the increase in intracellular temperature. R-CFPT has a functional advantage in that incorporation of R-CFPT into multiple living cells can be achieved without special devices. Therefore, it will be relatively easy to apply it to high-throughput screening of candidate materials to increase BAC thermogenesis. This method will be applicable to studies of the mechanism of thermogenesis of BACs and screening for a material that can elevate thermogenesis in the fight against obesity. BAC primary cell culture. Animal handling and primary cell culture was conducted in accordance with the guidelines for ethical animal care, handling, and termination of Kirin Company, which are in line with international and Japanese guidelines for animal care and welfare. This study was approved by the Kirin animal experiments committee. Three-week-old male Wistar rats were purchased from Charles River Japan (Tokyo, Japan). Isolation and culture of pre-BACs were performed as described 61 with modification. Briefly, a rat was sacrificed, and interscapular depots of brown fat were resected and cut into small pieces. The tissue fragments were shaken in Krebs-Ringer bicarbonate HEPES buffer containing 3 mg/ml of collagenase for 30 min at 37 °C. The digested tissue was filtered through a nylon screen with 100-μm pore size. The filtrate was centrifuged at 400 g for 5 min at room temperature. The pellets, which consisted of the stromal-vascular fraction (SVF) of the tissue, were suspended in "growth medium", comprising DMEM (4.5 g/L glucose, L-glutamine, sodium pyruvate) supplemented with 10% fetal calf serum (FCS), 17 μM D-pantothenic acid, 33 μM d-biotin, 100 μM ascorbic acid, 1 μM octanoic acid, 100 units/ml of penicillin, 0.1 mg/ml of streptomycin and 50 nM 3,3′,5-triiodo-L-thyronine (T3), and plated on 100-mm petri dishes. After incubation (5% CO 2 , 37 °C) for 3-4 days to give confluent pre-adipocytes (designated as "day 0"), differentiation was induced by replacing with fresh growth medium supplemented with 0.5 mM IBMX, 2.5 μM DEX, and 10 ng/ml of insulin. At day 2, the medium was changed to growth medium without FCS and supplemented with medium containing 0.1 nM insulin (maturation medium) to mature cells. Matured BACs were obtained on day 5 of incubation (5% CO 2 , 37 °C).

Materials
Quantitative real-time PCR. Total RNA was extracted from primary cultured BAC tissues with TRIzol reagent (Life Technologies) and purified using RNeasy (Qiagen, Hilden, Germany) in accordance with the manufacturers' instructions. cDNA was synthesized from total RNA by reverse transcription using the PrimeScript ™ RT Master Mix (Takara Bio, Shiga, Japan). Quantitative real-time PCR was performed with a LightCycler 480 instrument (Roche Diagnostics, Indianapolis, IN, USA) using SYBR Premix Ex Taq II (Takara Bio). Levels of mRNA were normalized to those of 18S rRNA. The sense and antisense primers are listed in Table 2.
Fluorescent polymeric thermometer. The R-CFPT used in this study was prepared as previously described 35 . Introduction of R-CFPT into pre-BACs and BACs. R-CFPT was incorporated into pre-BACs and BACs without cell detachment. SVF samples isolated from rat interscapular BAT were cultured on a collagen-coated To evaluate the efficiency of R-CFPT incorporation into a BAC, the thermometer was excited at 473 nm and the emission between 485 and 585 nm was monitored. To measure the temperature by ratiometric sensing, emission at 500-520 nm and 560-610 nm was monitored. The temperature of the culture medium was controlled using a microscope cage incubation chamber (Water Jacket Top Stage Incubator H101, Oko-Lab, Pozzuoli, Italy). In the Oko-lab H101 system, the temperature of the medium was monitored every minute with a type K thermocouple placed approximately 5 mm away from the microscopic field and was controlled by a feedback mode with a temperature accuracy of ±0.1 °C (Fig. S7 in Supplementary information). The chemical stimulus was applied by quickly adding 1 ml of pre-warmed chemical material in aqueous solution (culture medium containing 0.3% DMSO) to cells in 2 ml of culture medium. The temperature of the culture medium was maintained at 30 °C during analysis to perform the experiment within the linear response range of the calibration curve (as shown in Fig. 4a) and keep the temperature of the medium stable by reducing the deviation between room temperature and medium temperature.
The incorporation efficiency (%) was determined using Equation 2: BACs in DMEM were treated with 0.01 mg/ml of Hoechst33342 62 , 1 μg/ml of NileRed 63 , and 1 μM MitoTrackerDeepRed FM (Life Technologies) (final concentration) at 37 °C for 15 min to stain nuclei, lipid droplet, and mitochondria, respectively. After staining, R-CFPT was incorporated into cells. Hoechst33342 was excited with a 405-nm laser, and emission between 410 and 510 nm was monitored. NileRed was excited at 559 nm, and emission between 570 and 625 nm was monitored. MitoTrackerDeepRed FM was excited at 635 nm, and emission between 650 and 750 nm was monitored. For observation of R-CFPT, excitation was carried out at 473 nm and emission was monitored between 480 and 510 nm. Imaging analysis. Images were analyzed with Fluoview (Olympus) and MetaMorph (Molecular Devices, USA). Intracellular mapping as an intensity-modulated display (IMD) was performed with MetaMorph. A ratio image was created by dividing each pixel of two images after processing with a median filter and background subtraction. The intracellular temperature was calculated from the average value of the ratio image of a whole single cell based on the calibration curve. To show the difference between precursor cells and mature BACs more accurately and more simply, we analyzed the intracellular temperature change using the average signal of the whole cell. Analysis for intracellular temperature change was performed after excluding the cells in which the fluorescence intensity became half or less after the stimulation.
Measurement of the fluorescence response of R-CFPT in BAC extract. The fluorescence response of R-CFPT was evaluated in a BAC extract (0.01 w/v%) by confocal laser microscopy. The BAC extract was prepared as previously described 18 .
The temperature resolution (δT) of R-CFPT was evaluated using Equation 3 64 : where ∂T/∂F ratio and δF ratio represent the inverse of the slope of the fluorescence ratio-temperature diagram and the SD of the fluorescence ratio, respectively. The SD was obtained by triplicate measurements of one sample at each temperature.
Cell viability assay. A propidium iodide (PI) assay was used to determine cell viability, as described previously 35 . In brief, 0.5 mL of PI solution (2 μg/mL in PBS) was added to cells with introduced R-CFPT in 1 mL of FluoroBrite DMEM supplemented with 15 mM HEPES buffer, and cells were cultured at 37 °C for 30 min. Fluorescence images of PI were acquired by exciting the cells at 559 nm and recording the emission between 655 and 755 nm using the variable bandpass filter sets of a DM405/473/559 excitation dichroic mirror. Cell viability (%) was determined using Equation 4: = ×