Interleukin 6 (il 6)

Concentrations of MMP1, MMP3, MMP13, IL6 (Cloud Clone Corp, Katy, Texas, USA) and ADAMTS5 (MyBioSource, San Diego, California, USA) in the cell culture supernatant were determined using commercially available ELISA kits according to the instructions provided by the manufacturer. Values are presented in pg/mL. A standard curve was calculated for each assay with the following detection limits: MMP1 < 0.134ng/mL, MMP3 < 12.9pg/mL, MMP13 < 0.29ng/mL, ADAMTS5 = 31.2ng/mL and IL-6 <3pg/ml.

Serum levels of IDO and cytokines (TNF-α, IL-1β, IFN-γ, IL-4, IL-6, IL-17) were determined at baseline and at 6 weeks of follow-up. Venous blood was collected from the forearm between 6 and 7 a.m. following an overnight fast. Serum levels of IDO and six cytokines were measured by quantitative enzyme-linked immunosorbent assay (ELISA) using a commercially available kit: IDO (SEB547Hu, sensitivity: minimum detectable measurement ≤ 0.118 ng/mL) (Cloud-Clone, Wuhan, China), IFN-γ (SEA049Hu, sensitivity: minimum detectable measurement ≤ 6.1 pg/ml) (Cloud-Clone, Wuhan, China), IL-4 (SEA077Hu, sensitivity: minimum detectable measurement≤5.9 pg/ml) (Cloud-Clone, Wuhan, China), IL-6 (SEA079Hu, sensitivity: minimum detectable measurement ≤ 3.2 pg/ml) (Cloud-Clone, Wuhan, China), and IL-17 (SEA063Hu, sensitivity: 5.5 pg/ml) (Cloud-Clone, Wuhan, China), TNF-α (DTA00D, sensitivity: 6.23 pg/ml) (R&D Systems, USA), IL-1β (DLB50, sensitivity: 1 pg/ml) (R&D Systems, USA). All ELISA kits used in this study have been cross-reactivity validation of existing related factors to ensure their specificity. All assays were blinded to group or behavior information until after the results were finalized.

We performed evaluations on soluble factors from supernatants of uncultured mSVF and eSVF through the commercially enzyme-linked immunosorbent tests (ELISA). To this end, mSVF and eSVF were centrifuged at 600 g for 10 min, and their supernatants were collected to test the released cytokines. Moreover, we plated and cultured mSVF and eSVF to test whether the in vitro culture can modify their cytokine profile compared to the native uncultured adipose products. After 80% confluence, culture supernatants (S/N) from both mSVF (mSVF S/N) and eSVF (eSVF S/N) were recovered and kept in serum-free medium for 48 h before their collection. We performed ELISA assessments on the following molecules: vascular endothelial growth factor (VEGF) (Cloud-Clone Corp, Texas, US), Platelet-derived growth factor (PDGF)-bb (LifeSpan Biosciences, Washington, US), insulin growth factor (IGF)-1 (Cloud-Clone Corp), hepatocyte growth factor (HGF) (Cloud-Clone Corp), interleukin (IL)-10 (Cloud-Clone Corp), interleukin (IL)-1β (Cloud-Clone Corp), IL-6 (Cloud-Clone Corp), IL-21 (MyBioSource, California, US), IL-23 (MyBioSource), tumour necrosis factor (TNF)-α (Cusabio, Houston, Texas, US). We used the manufacturer’s instructions through spectrophotometer measurement at a wavelength of 510–570 nm.

Serums were from fasting blood samples. The levels of TNF-α and IL-6 were performed as per the manufacturer's instructions (Cloud-Clone Corp., Houston, USA), and absorbance kinetics was measured through an ELISA reader.

Clobenpropit hydrobromide (CLO) was purchased from Cayman Chemical (Ann Arbor, MI, USA) and lipopolysaccharides (LPS) from Escherichia coli were procured from Sigma-Aldrich Co (St. Louis, MO, USA). Mouse TNF-α, IL-6, TGF-β1, IL-10, and COX- 2 were purchased from Cloud-Clone Corp., Houston, TX, USA. Mouse MRCC-I, MRCC-II, and MRCC-IV were purchased from MyBioSource, Inc., San Diego, CA, USA.