Asys uvm 340 spectrophotometer

Proteins in approximately 1 mL of apoplastic fluid were precipitated by adding five volumes of cold 10% TCA. Samples were incubated for at least 14 h at 4°C and then centrifuged at 10,000 g for 15 min. The pellet was washed twice with cold methanol, dried with N₂ gas and solubilized in a sample rehydration buffer containing 8 M urea, 2% (w/v) CHAPS, 50 mM DTT, 2 mM PMSF and 0.2% (v/v) IPG buffer pH 3–10 (GE Healthcare, Uppsala, Sweden). After rehydration, samples were incubated in a Thermomixer Confort device (Eppendorf AG, Hamburg, Germany) at 29°C and 1000 rpm during 3 h, then centrifuged at 10,000 ×g for 10 min at RT and filtered (0.45 μm ultrafree-MC filters, Millipore, Bedford, USA). Protein concentration in the samples was quantified immediately with the Bradford method in microtiter plates using an Asys UVM 340 spectrophotometer (Biochrom Ltd., Cambridge, UK) and BSA as standard.

4T1 cells were seeded in sextuplicate into 96-well plates at a density of 5000 cells per well in 100 μL of medium and allowed to attach overnight. Cells were treated with increasing concentrations of E.A. and CDDP for 24, 48, and 72 h or their combinations for 48 h. At the end of treatment, cells were washed with phosphate-buffered saline (PBS), stained for 5 min with a crystal violet solution (0.5% (w/v) crystal violet in 25% (v/v) methanol), and then gently rinsed with water. Absorbances were read at 540 nm with a Biochrom Asys UVM 340 spectrophotometer following extraction of the dye by 0.1 M sodium citrate in 50% ethanol. The inhibitory concentrations of 50% (IC₅₀) were calculated using a four-parameter nonlinear regression with GraphPad Prism version 6 software (GraphPad software, La Jolla California USA).

Serum proteins (FBS, Gibco) were diluted (1:5) in phosphate-buffered saline (PBS), adsorbed to flat PCU and CNT/PCU composites of varying ratios, and stored for 4 hrs in a cell culture incubator to emulate the cell adhesion environment. Then, protein solutions were removed using a solution of 2% (w/v) sodium dodecyl sulfate (SDS; L3771, Sigma). The concentration of proteins was determined with a Coomassie Plus (Bradford) Assay Kit (23236, Sigma). The absorbance at 595 nm was measured using an Asys UVM 340 spectrophotometer (Biochrom). Protein concentrations were determined by extrapolation from a standard curve for albumin, and were normalized to glass.
Human vitronectin (VN; V8379, Sigma) adsorbed on PCU, 10% CNT/PCU, and 50% CNT/PCU surfaces under standard cell culture conditions were used to evaluate VN adsorption. After 4 hrs, VN was removed with 2% (w/v) SDS (L3771, Sigma) and surfaces gently washed three times. The adsorbed VN on sample surfaces was measured with a Coomassie Plus (Bradford) Assay Kit (23236, Thermo) at 595 nm using an Asys UVM 340 spectrophotometer. The amount of VN in solution was calculated by extrapolation from a standard curve generated for VN.