2.5.2. Trypan Blue Assay

The MTT assay detects viable cells but does not take into consideration cell loss caused by cell death; thus, the percentage of viable cells was additionally determined using a dye exclusion test with trypan blue (TB) dye, which is based on the principle that living cells possess intact cell membranes that exclude TB, whereas dead cells do not. The TB test was performed as previously described [41]. Briefly, an aliquot of cell suspension was centrifuged (950 rpm during 5 min, Eppendorf Centrifuge 5804, Hamburg, Germany), and the pellet was resuspended in PBS or serum-free complete medium. Then, 10 μL of cell suspension corresponding to each experimental condition was mixed with 10 μL of TB (0.4%). After incubating the mixture for 3 min at room temperature, the cells were examined using an automated cell counter (TC10TM, Hercules, CA, USA, BioRad).

### *2.6. Expression of Main Genes Related to Early and Late Di*ff*erentiation*

To analyze the expression of early and late genes, cells were collected at the ID and FD time-points, respectively. Total RNA was extracted from cells di fferentiated in sterile six-well plates (at a cellular density of 8 × 10<sup>4</sup> cell/well) using the extraction kit PureLink ™ RNA Mini Kit (Waltham, MA USA, ThermoFisher Scientific), according to the manufacturer's instructions. The extracted RNA was verified and quantified spectrophotometrically using NanoDrop (Thermo Scientific). Complementary DNA (cDNA) was synthesized from 1 μg of RNA using the RevertAid H Minus First-Strand cDNA synthesis kit (Fisher Scientific), according to the manufacturer's protocol. Gene expression was assessed using quantitative real-time PCR (qRT-PCR) in a LightCycler 480 II thermocycler with Fast Sybr Green Master Mix (Waltham, MA USA, Applied Biosystem). β-Actin was used as an endogenous control. The primer sequences used for amplification are presented in Table A1 (Appendix A).

The reaction mixtures were incubated for an initial denaturation at 95 ◦C for 10 min, followed by 45 PCR cycles (95 ◦C for 15 s, 60 ◦C for 1 min, 95 ◦C for 15 s, and 60 ◦C for 1 min). The ΔΔCT method was used to measure relative quantification, and the levels of transcripts were normalized to that of β-actin. The levels of each mRNA were calculated as relative expression to the basal condition (designated as 1). 3T3-L1 cells treated for 48 h with the AC, without CCT, were considered representative of the basal condition. Three independent experiments were performed, each in duplicate.

Calibrated ΔCt values from undi fferentiated and control di fferentiated cells were used to evaluate the expression of genes. Fold changes in gene expression were calculated using the 2−ΔΔCt method [28]. Expression of the *aP2-1* gene was used as a specific adipocyte marker. *aP2* is widely used as a marker of di fferentiated adipocytes [42].
