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in dimethyl sulfoxide for 30 min at 37°C and the absorbance was determined at 570 nm with a microplate reader (Tecan, Mannedorf, Austria). Celi proliferation was expressed as percent of absorbance vs values of day 0.

3.5.6 PCR

For gene expression analysis, bonę marrow MSCs from WT and Cd36-nuli mice were seeded in 60-mm culture dishes and incubated for 7 days. For Wnt3a stimulation assays, the MSCs from WT and Cd36-m\\ mice were cultured in a-MEM medium containing 20% FBS for an additional 24 h without or with 100 ng/mL Wnt3a (Sigma-Aldrich, Oakville, Ontario, Canada). Total RNA was extracted from cells using TriZol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription (RT) reactions were carried out with Omniscript RT kit (Qiagen, Mississauga, Ontario, Canada) using hexamers as random primers. PCR amplifications for the genes of interest were conducted using specific primer sets (Tab. 3.1). Real-time PCR analysis was performed using the iCycler IQ detection system (Bio-Rad, Hercules, CA, USA) and SYBR Green I (Takara, Mountain View, CA, USA) as a double-strand DNA-speciflc binding dye, using (3-microglobulinę as reference gene. Each sample was run in triplicate, and fluorescence data were collected at the end of the extension step in every cycle. To ensure specific amplification, a melting curve was calculated for each PCR reaction by increasing the temperaturę from 60 to 95°C with a temperaturę increment ratę of 0.5°C/10 seconds. Fold induction and expression levels for all gene were calculated using the comparative CT method [i.e., 1/(2ACT), where ACT is the difference between CT target and CT reference] after normalization to (3-microglobuline expression level, and data were analyzed using optical system software Yersion 3.1 (Bio-Rad).