Mem nonessential amino acids 100x

To promote the formation of tissues, bYS-MSCs (25 to 34 days of gestation) were cultured in α-MEM culture medium (minimum essential medium, Gibco) supplemented with 10% fetal bovine serum (Gibco), BME amino acid solution 50x (Sigma-Aldrich), MEM nonessential amino acids 100x (Sigma-Aldrich), β-mercaptoethanol solution 100x (Gibco), and a 1% v/v antibiotic solution (penicillin G 10.000 units/mL and streptomycin 10.000 μg/mL) and were trypsinized (TrypLE Express; Gibco-BRL). When they reached 80% confluence, they were replaced. From passage 4, the cells started an aggregation process to spontaneously form a three-dimensional tubular structure. To evaluate the tubular structure, histology, transmission electron microscopy, and electronic microscopy techniques were performed.

The following were purchased from Sigma-Aldrich (Dorset, UK): Dulbecco’s Modified Eagle Medium (DMEM), Penicillin/Streptomycin, MEM non-essential amino acids 100x, Trypsin-EDTA, L-glutamine. Foetal bovine serum (FBS) was from Gibco Life Technologies, Sao Paolo, Brazil. The protease and phosphatase inhibitors cocktail were from Roche, Indianapolis, IN, USA. The following antibodies were purchased from Abcam Biotechnology, Cambridge, UK: rabbit monoclonal [EPR26155-110-1] to β-catenin, rabbit monoclonal [EPR21889] to WNT-3a, rabbit monoclonal [E87] to Caspase-3, and rabbit polyclonal to GAPDH—the loading control. Curcumin, resveratrol, and Doxorubicin were purchased from Sigma-Aldrich, St. Louis, MO, USA.

YS cells were cultured following the protocol of Mançanares et al. [1 (link)]. Cells were first washed in a solution of PBS-L (without calcium or magnesium) supplemented with antibiotics (5% penicillin-streptomycin; Invitrogen, Carlsbad, CA, USA) and then macerated enzymatically with 0.5% collagenase IV (Sigma-Aldrich) for 1 hour. The collagenase was inactivated with α-MEM culture medium (minimum essential medium, Gibco) supplemented with 10% fetal bovine serum (Gibco), BME amino acid solution 50x (Sigma-Aldrich), MEM nonessential amino acids 100x (Sigma-Aldrich), β-mercaptoethanol solution 100x (Gibco), and a 1% v/v antibiotic solution (penicillin G 10.000 units/mL and streptomycin 10.000μg/mL). Cells were plated in 25 cm² plates in a 37°C incubator with 5% CO₂.