Ec plan neofluar 20x 0.5 objective

Adipogenic medium consisted of DMEM/F-12 supplemented with 10% FBS, penicillinstreptomycin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich), 10 µM dexamethasone (Sigma-Aldrich), 66 µM indomethacin (Sigma-Aldrich), 2 µL/mL insulin (insulin lispro injection, 100 U/mL, Humalog ® ) [20] . MSCs were maintained in adipogenic medium for 3 days, detached with 0.05 % trypsin-EDTA, fixed with 4% paraformaldehyde (Sigma-Aldrich) and lipid droplets were stained with fluorescent AdipoRed™ according to the manufacturer's instructions (Lonza). Fluorescent lipid droplets were detected with Axioskop 2 Mot fluorescence microscope (Carl Zeiss) using EC Plan-Neofluar 20x/0.5 objective (Carl Zeiss). For quantitation of lipid accumulation in adipocytes, the cells (5 × 10 5 cells/sample) were labeled with AdipoRed™ as described above and quantified by FACSCalibur flow cytometer using CellQuest TM software.
Osteogenic medium was composed of CM and 10 mM β-glycerophosphate (Sigma-Aldrich), 50 µg/mL L-ascorbic acid 2-phosphate (Sigma-Aldrich) and 0.1 µM hydrocortisone (Sigma-Aldrich). MSCs were cultured in osteogenic medium for 14 days then fixed with 8% formaldehyde and stained with Alizarin Red S (Sigma-Aldrich) solution (pH 4.1) to detect calcium deposition. Photomicrographs were taken with Olympus CKX41 inverted light microscope and Olympus Camedia C-5060 camera (Olympus Holding Europa GmbH).