Dopamine solution

A microdialysis probe (1mm CMA-7, 6 kDa, CMA) connected to an infusion pump was inserted through the cannula placed in the striatum. Artificial cerebrospinal fluid (ACSF) was perfused at 1.2 μL/min. After the probe insertion we waited for 40 minutes to avoid artifacts evoked by mechanical manipulation. Each sample was collected for 5 minutes in awake animals moving freely on a custom designed jetball system. Immediately after collection, each sample was quantified by an HPLC system (Eicom). Chromatograms were analyzed with the software EPC-300 (Eicom). Dopamine concentration was determined using a dopamine solution of 0.5 pg/μL (Sigma-Aldrich). The temporal course of uncaged dopamine was normalized to the maximum peak evoked by LED irradiation. We used such normalization because the measurements of uncaged dopamine in the samples varied as a function of the distance between the fiber optic and the microdialysis probe.

PDMS substrates were prepared by mixing ten parts of silicone elastomer base with one part of curing agent (SYLGARD, Dow Corning, USA) and stirred. The mixture was then poured into well plates or culture dishes, degassed for 30 min in a vacuum oven to remove air bubbles, followed by heat-curing at 70 °C for 100 min. Surface modification was performed by immersing the native PDMS surface in dopamine solution (Sigma Aldrich, Singapore) prepared in 10 mM Tris-HCl (pH 8.5). PD concentration (0.000%w/v–0.100%w/v) and duration of coating (0–24 h) were varied to investigate the effect of these parameters on the stability of BMSC adhesion and proliferation. After PD coating, the surfaces were rinsed twice with 1X Phosphate Buffered Saline (PBS) to remove unattached PD molecules. Additional coating of 20 μg/ml collagen type 1 (Life Technologies, Singapore) was performed to evaluate the combinatorial effect of PD and collagen on BMSC behaviour. Lastly, the uncoated and PD-coated PDMS substrates were UV-sterilized for 1 h prior to cell culture.

Polyethylene membranes with a diameter of 45 mm were polished with 1500-grit silicon carbide paper and then ultrasonically cleaned with deionized water. The polished polyethylene membranes were immersed in a freshly prepared 2 mg/mL dopamine solution (Sigma-Aldrich, St. Louis, MO, USA) (10 mM Tris buffer, pH 8.5) at 37 °C in the dark. After immersion for 24 h, the membranes were cleaned ultrasonically for 10 min with deionized water three times and dried under nitrogen.