## 4 Discussion  
Obesity is a state in which adipocytes in the body are abnormally accumulated. Obesity generates various social, economic, and medical issues, and is possibly related to the development of various human diseases, such as cardiovascular disease, type II diabetes, infertility, osteoarthritis, and certain cancers [7; 8]. Recently, concerning the problem of obesity, research on brown fat cells or browning has been actively conducted [14; 15]. Brown fat cells are cells that consume a lot of energy as heat energy due to non-shivering thermogenesis mediated by UCP1 of a large number of mitochondria. In terms of brown fat cells, a previous study discussed the involvement of pannexin-1 channels in adaptive thermogenesis in brown adipose tissue [16]. In addition, flap necrosis in reconstructive surgery is a common surgical complication, and to treat this, adipose-derived stem cells are used. Like this report, the use of adipose-derived stem cells is increasing [17]. If a promising mechanism of browning is discovered in the future, it will cause a drastic change in the treatment of obesity. Thus, this study investigated whether the FTIR system can be applied for characterizing adipose cells. The differentiation of adipocytes for this study was confirmed through Oil-red-O staining and the presence of the following markers: white adipocyte markers (PPARy, C/EBP\(\alpha\), aP2) and brown adipocyte markers (PRDM16, PPAR\(\gamma\), PGC-1\(\alpha\), UCP1). PPAR\(\gamma\) is a key regulator of adipocyte differentiation and is an important transcription factor for adipocyte metabolism and differentiation [14]. C/EBP\(\alpha\) directly leads to lots of adipocyte genes [14]. UCP1 is expressed distinctly in brown adipocytes as heat without generating ATP. PGC-1\(\alpha\) is a marker that leads to mitochondrial biogenesis and UCP1 expression [18]. PRDM16 is a marker to control the fate of brown adipocytes [19]. Since Oil-red-O staining and the above specific markers confirmed the identity of adipocytes (Figure 1A,B), these adipocytes were used as a sample for the next experiment. In FTIR comparison with conditioned media of adipocytes (Figure 2A,B and Supplementary Figure S1A,B), amide I/amide II ratios were different between MBCM (2.42) and BPCM (3.96) (Figure 3A) [20; 21]. It is believed that high amide I/amide II ratios affect protein pattern composition [22]. BPCM had the highest protein content (0.79) (Figure 3B) [20; 21]. Therefore, the differentiation process of brown adipocytes is presumed that the function of secretory protein affects differentiation differently from that of white adipocyte differentiation. In FTIR comparison with adipocytes (Figure 4A,B and Supplementary Figure S1C,D), various chemical constituents such as lipid amount, amide I/amide II ratio, and lipid acyl chain length in adipocyte types were analyzed to determine any difference [20; 21]. It was confirmed that differentiated adipocytes contained more fat mass than progenitor cells (Figure 5A) [20; 21]. Unlike in conditioned media, amide I/amide II ratios were similar in the cells, showing slightly higher from WPA and WAT (Figure 5B) [20; 21]. Because amide absorption is sensitive to protein formation, the amide I/amide I + amide II ratio is an indicator of protein level [20]. Protein rate is an important aspect of cell regulation during differentiation [23; 24]. Therefore, there is a less significant change in the composition and content of intracellular proteins, but it can be deduced that small differences affect the differences among cells, especially in the differentiation of white adipocytes. WAT showed the longest lipid acyl chain lengths (Figure 5C) [20; 21]. Lipid acyl chain length has important effects on cholesterol orientation in monousaturated bilayers [25]. This phenomenon was inferred to the functional difference between WAT and BAT. This is the first study to examine the properties of adipocyte-conditioned medium and adipocytes at different stages of development using FTIR spectroscopy.