Three way stopcock

The study was approved by The Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China (SH9H-2018-T22-1). After obtaining consent forms, adipose tissue was harvested from the abdomen or thigh of healthy adult females using liposuction from December 2020 to November 2021. In this study, CEFFE was derived from 5 females aged from 22 to 35. The preparation procedure of CEFFE followed previously established protocol [24 (link)]. Briefly, the harvested adipose tissue was flushed with physiological saline and centrifuged at 1200 g/4 °C for 3 min 2 or 3 times to remove tissue debris, blood, and oil. Then, the emulsified fat was obtained by mechanical emulsification through two 10 mL syringes (KDL, China) connected by a three-way stopcock with an internal diameter of 2 mm (Terumo Corporation, Japan) 60 times. Immediately after, the emulsified fat was centrifuged at 1200 g/4 °C for 5 min, and the liquid in the bottom was withdrawn. Finally, the extract supernatant was filtrated with a 0.22 μm filter (Corning, USA) as CEFFE and subsequently frozen at − 80 °C for future experiments [24 (link)]. The protein concentration in CEFFE was determined with a bicinchoninic acid assay kit (BCA, Thermo Fisher Scientific, USA).