Synergy 4 spectrophotometer

VEGF released from SIS was assessed using in-direct ELISA. Buffers used for release kinetics include PBS, PBS + 196U/mL heparin, PBS+ 1960U/mL heparin, and growth media (EGM2; Lonza) + 10% FBS using SIS pieces with and without immobilized heparin followed by VEGF immobilization. SIS pieces (n=8 per condition) were incubated at 37°C and 10% CO₂ for a total of 120hr. At time points of 24, 48, 72, and 120 hr the buffer was replaced with fresh buffer and analyzed for VEGF concentration using ELISA (VEGF Duoset, R&D Systems) according to the manufacturer’s instructions. Absorbance was read at 450nm using a Biotek Synergy 4 Spectrophotometer (with subtraction of background absorbance of 570nm). Cumulative amount of VEGF was calculated over time and normalized to the initial amount of immobilized VEGF.

The determination of COX-1 and COX-2 enzyme inhibitory activity was performed using the kit supplied by Cayman Chemical (Ann Arbor, MI, USA). The kit depends on assaying the peroxidase activity of the enzymes and the colorimetric monitoring of the appearance of oxidized N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) at 590 nm. In a transparent 96-well plate the enzymes [COX-1 (bovine) and COX-2 (human)] were diluted in the assay buffer supplied with the kit. The enzymes were then mixed with heme and the inhibitors (dissolved in DMSO at the required concentrations) and incubated for 5 min at 25 °C. The colorimetric substrate was then added to the reaction wells and the reaction was directly initiated by the addition of arachidonic acid. After 2 min the plate was read using a BioTek Synergy 4 spectrophotometer at 590 nm. The IC₅₀ values were determined by plotting the % inhibition of the enzyme activity against the inhibitors concentration. The data presented are the mean of three different experiments.

Laminated SIS tubes (4cm in length and 4.5–4.75mm diameter) were provided by Cook-Biotech Inc (West Lafayette, IN). For heparin immobilization, SIS was immersed in 10mL of 2-(N-morpholino)ethanesulfonic acid buffer (MES), pH 1.5, containing 20mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 20mM N-Hydroxysuccinimide (NHS), and heparin sulfate (8000U) and gently agitated for 36hr at 37°C. SIS was then gently washed in PBS to neutralize the reaction and remove unbound heparin. Heparin concentration on SIS was determined by comparing the unbound heparin concentration remaining in solution to the initial concentration [34 (link)]. Briefly, unbound heparin was added to toluidine blue buffer (0.1mg toluidine blue dissolved in 50mM tris-buffered saline (TBS) buffer, pH 4.5) at a ratio of 1 volume toluidine blue to 12 volumes heparin solution. The reaction proceeded for 30min prior to centrifugation and absorbance was measured at 631nm using a Biotek Synergy 4 Spectrophotometer (background absorbance of an empty well was subtracted from each value). To immobilize VEGF, heparin bound SIS was then immersed in 10mL of PBS with 3mg of recombinant human VEGF-165 for 8hr, washed with PBS, and either immediately implanted or analyzed. VEGF remained immobilized on the heparin surface by utilizing the heparin binding domain[35 (link)].