Coomassie plus bradford assay kit

Ten micrograms of LPPC were incubated with different amounts of OKT3 or 2C11 for 20 min and centrifuged at 5,900 × g for 5 min. The pellets were resuspended, and the quantity of OKT3 or 2C11 bound to LPPC was determined by using a Coomassie Plus Bradford Assay Kit (Sigma‒Aldrich) according to the standard procedures provided by the manufacturer. The supernatants were analyzed as described above.
Peripheral blood mononuclear cells (PBMCs) were seeded in a 96-well plate at 1 × 10⁵ per well in 100 µl RPMI medium containing 10% FBS and 1% PSA. LPPCs (1µγ) with different amounts of mAbs were added respectively to each well of a 96-well plate. After incubation for three days, the cell proliferation rates were determined by the MTT assay (Sigma‒Aldrich).
Similarly, in the naive group, splenocytes (2.5 × 10⁵ cells, each well of a 96-well plate) from naive mice were incubated with 1 µg LPPC with different amounts of mAbs for three days. In the activated groups, splenocytes from naive mice were activated by anti-CD3 (60 ng/100 µl) and anti-CD28 mAbs (60 ng/100 µl) for three days. After stimulation, the splenocytes were reseeded into a 96-well plate. The cell proliferation rate was monitored by MTT assay.

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.