Cfx96 system cycler

Cells were kept overnight in starvation medium (α-MEM (11095-080, Gibco) containing 0.5% FBS (10270-106, Gibco) and 1% Penicillin/Streptomycin (15140-122, Gibco)). Further, 100 ng/ml Adiponectin (50636-M08H, Sino Biological Inc.) were added for indicated time periods: 0, 45 min, 1.5 and 3 h. Then, total RNA was isolated using the NucleoSpin^® RNA Kit (740955.50, MACHEREY-NAGEL). RNA concentrations were measured at 260 nm using a spectrophotometer (Nanodrop2000, Thermo Scientific). cDNA was synthesized from 1.0 μg of total RNA using the InnuSCRIPT Reverse Transcriptase kit (845-RT-6000100, Analytik Jena) and performed on CFX96^TM System Cycler (Bio-Rad).
The SsoAdvanced^TM Universal SYBR^@ Green Supermix (1723271, Bio-Rad) was used in each reaction setup. The primers employed were: Mouse AdipoR1&2 (qMmuCID0023619, qMmuCID0010157), AP (qMmuCED0003797), BSP (qMmuCID0006396), OCN (qMmuCED0041364), OPG (qMmuCID0005205), Runx-2 (qMmuCID0005205) and F-Spondin (qMmuCED0049433) all from Bio-Rad. GAPDH (qMmuCED0027497, Bio-Rad) was used as housekeeping gene. Results were analyzed using the Bio-Rad CFX Manager 3.1 software.

Total RNA was extracted using the NucleoSpin^@ RNA Kit (740955.50, MACHEREY‐NAGEL). The quality and quantity of the eluted mRNA were measured for optical density photometrically using a spectrophotometer (Nanodrop2000, Thermo Scientific). cDNA was synthesized from 1.0 μg of total RNA using the iScript^TM cDNA Synthesis Kit (#1708891, Bio‐Rad) and performed in the CFX96^TM System Cycler (Bio‐Rad).

The total RNA was isolated using the ReliaPrep™ RNA Miniprep System (z6011, Promega, Madison, WI, USA). RNA concentrations were measured at 260 nm using a spectrophotometer (Nanodrop2000, Thermo Scientific, Waltham, MA, USA). cDNA was synthesized from 1.0 μg of total RNA using the Verso cDNA Synthesis Kit (AB1453B, Thermo Fisher, Waltham, MA, USA) and synthesis was performed using a CFX96TM System Cycler (Bio-Rad, Hercules, CA, USA).
The SsoAdvancedTM Universal SYBR@ Green Supermix (1723271, Bio-Rad, Hercules, CA, USA) was used in each reaction setup. The primers employed were mouse inducible nitric oxide synthases (Nos2) (QT01547980, Qiagen, Venlo, Limburg, The Netherland), interleukin 1β (Il1b) (QT01048355, Qiagen), Arginase 1 (Arg1) (QT00134288, Qiagen), interleukin 10 (Il10) (QT00106169, Qiagen), Saa3 (QT00249823, Qiagen), and ApoE (QT01043889, Qiagen). β-Actin (QT00095242, Qiagen) was used as a housekeeping gene. Results were analyzed using Bio-Rad CFX Manager 3.1 software.