## 4 Discussion
Based on the findings obtained in this study, it is expected that when a cell-conditioned medium without preparation is analyzed by an infrared microscope with high resolution, it will be possible to perform metabolic studies, structural analysis, and protein changes such as alpha-helix and beta-sheet of proteins released in the differentiation process of adipocytes [26]. Various adipokines, for example, have been discovered through the investigation of variations in protein secretion during adipocyte development and a difference in the secreted proteins was noted during the differentiation process. If infrared analysis withhigh resolution would be applied, it would be possible to expand the research of secretory proteins [27]. When obesity-related diseases are recognized as global issues, attention is focused on beige adipocytes. The differences in the expression of the Hox family were raised in this process [28; 29; 30; 31]. In studies, when a deep understanding of the cell is needed, the application of infrared microscope with high resolution is required to provide cell-related chemical information by the non-invasive method. Therefore, this paper presented the future direction of adipocyte characterization studies using advanced FTIR spectroscopy as above and is expected to contribute information in research using FTIR when searching for obesity-related important metabolites and anti-obesity drug candidates. Furthermore, cell differences between 3000 and 2840 and between 1740 and 1340 cm\({}^{-1}\) of the pre-treated spectra were observed (Supplementary Figure S2). FTIR spectra showed the difference of WPA, WAT, BPA, and BAT at two important regions, both the lipid region and the protein region. In this regard, further research through molecular biological analysis in connection with FTIR is likely to be needed. These spectral differences enabled the discrimination of adipocytes when applying principal component analysis. It was found that mature adipocytes were widely distributed, which was presumably due to cell differentiation (Figure 6A,B). Additionally, cluster by dendrogram was tested but was less than PCA (Supplementary Figure S3). Currently, there is a growing need for research on adipose-derived stem cells, whereby a method for selectively screening only adipose-derived stem cells from adipose tissue is necessary. FACS, which is currently used as a cell sorter, basically requires tagging of cells. However, the cell labelling process is not necessary for an infrared microscope system, making it possible to develop an inexpensive and efficient device in connection with a kind of microfluidic cell sorter. In this way, we presented a brand-new cell sorter using FTIR spectroscopy. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12063071/s1](https://www.mdpi.com/article/10.3390/app12063071/s1)]([https://www.mdpi.com/article/10.3390/app12063071/s1](https://www.mdpi.com/article/10.3390/app12063071/s1)), Figure S1: Raw spectra of conditioned media [WAM (black), WPCM (red), MWCM (blue), BAM (green), BPCM (yellow), MBCM (orange)] and adipocytes [WPA (black), WAT (red), BPA (blue), BAT (green)]. (A) Full-range scale spectra of adipocyte conditioned media (B) Adipocyte conditioned media' spectra between 1850 cm\({}^{-1}\) and 1340 cm\({}^{-1}\) (C) Full-range scale spectra of adipocytes (D) Adipocytes' spectra between 1850 cm\({}^{-1}\) and 1360 cm\({}^{-1}\) marked with positions of 1640 cm\({}^{-1}\) (peak suspected by the effect of water) and 1634 cm\({}^{-1}\) (water peak), Figure S2: Spectra used in PCA for discrimination of adipocytes, Figure S3: Cluster by dendrogram for discrimination of adipocytes. Conceptualization, D.S. and S.P. ; methodology, D.S. and S.P. ; software, D.S. ; validation, D.S., S.P., S.Y. and Y.K. ; formal analysis, D.S. and S.P. ; investigation, D.S. and S.P. ; resources, D.S. and S.P. ; data curation, D.S. and S.P. ; writing--original draft preparation, D.S. and S.P. ; writing--review and editing, Y.K. ; visualization, D.S. and S.P.