Millex gp syringe

When FDMSCs and ADFs reached 80% confluence in 12-well plates (Corning, USA), cells were cultured in serum-free medium (SFM) overnight. Then cells were cultured with 1 ml SFM per well. F-CM and A-CM were harvested after 48 h incubation. The conditioned medium was then filter-sterilized through a 0.22-μm Millex-GP syringe filter (Millipore, USA) and stored at –80 °C.

To make the first stock solution of the CAs, autoclaved, ultrapure water (MilliQ Purification System, Millipore, Darmstadt, Germany) was added to MnTPPS₄ (5,10,15,20-Tetrakis(4-sulfonatophenyl)-21H,23H-porphine manganese(III) chloride, Sigma-Aldrich Canada Co., Oakville ON, Product# 441813) and MnTPPS₃NH₂ (synthesized in our laboratory). The CA solutions were filtered using a 33-mm-diameter sterile Millex-GP syringe filter unit with a 0.22-μm pore size hydrophilic polyethersulfone membrane (SLGP033RS, Millipore, Darmstadt, Germany). The first stock solutions were sono-bathed in an ultrasonic cleaner (VWR symphony, VWR International, Mississauga, Canada) for 30 minutes at about 65 ^oC. To further sterilize and solubilize the stock solutions, they were placed at a hotplate, preheated at 100–200 ^oC, for approximately 5–7 minutes. Serial dilutions (1000X) were made inside a biosafety cabinet in order to decrease the concentrations of the sterilized first stock solutions of the CAs to detectable levels on a UV-visible spectrophotometer. The concentrations of the diluted solutions were then determined by measuring absorbance using a UV-visible spectrophotometer. Knowing the dilution factors, the concentrations of the first stock solutions of the CAs were found.