2.7. Analysis of gene expression by RT-qPCR
Total RNA was extracted from liver and fat samples using TRIzol® Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), following the manufacturer’s recommendations, and was reverse transcribed into cDNA using oligo(dT) primers (Promega, Southampton, UK).  Real time quantitative PCR (qPCR) amplification and detection was carried out on optical-grade 48 well plates in EcoTM Real time PCR System (Illumina, San Diego, CA, USA) with 20 ng of cDNA, the KAPA SYBR® FAST qPCR Master Mix (Kapa Biosystems, Wilmington, MA, USA) with specific primers (Table 1). The mRNA relative quantitation was calculated using the ΔΔCt method and glyceraldehyde 3-phosphate dehydrogenase (Gapdh ) was used as housekeeping gene.
2.8. Flow cytometry
The cells from adipose and liver tissues were collected following the procedure previously described with some modifications(Anderson, Carrillo-Gálvez, & Martín, 2015). CD45+CD11b+ and CD11b+Ly6C+