Cells seeded in a 96-well microplate (Corning, NY, USA) at a density of 2 × 10 cells/well were cultured with or without IL-1β (5 ng/ml) Sigma-Aldrich, Saint Louiss, Missouri, USA).
After 5 to 6 days, the cells were seeded onto 0.33-cm2 0.33-cm2-coated transwell filters (0.4-μm pore size, collagen-coatedTranswell Collagen T-cols, Acton, MA, USA) at a density ofiltersximately 2 × 106/cm2.
Cells were then seeded onto the autoclaved titanium samples placed in a 12-well culture plate (Falcon, BD Biosciences, San Jose, CA, USA) at a density of 5 × 103 cells/cm2 for 3 days for cell adhesion assay and 1 × 104 cells/cm2 for 1 week for cell proliferation assay, respectively.
The NIH/3T3 cells were trypsinized and then seeded into polystyrene 96-well plates (Nalge Nunc International, Penfield, NY, USA) at a density of 3 × 104 cells/mL and then after 24 h, the cultured cells were incubated with NO/THCPSi NPs, glucose/THCPSi NPs, and THCPSi NPs at four different concentrations from 0.05 to 0.2 mg/mL for 48 h.
For live cell imaging, cells were plated on chambered cover glasses (Nunc, Rochester, NY, USA) at a density of 80,000 cells per chamber in 2 ml medium.
After mechanical dissociation, centrifugation, and resuspension, the cells were seeded in 6-well or 24-well tissue culture plates (Coning, USA) pre-coated overnight with poly-L-lysine (Sigma, USA)) at a density of 1 × 105/cm2.
Equal numbers of parental BC/BY cells or activated test cells BCS/BYA were mixed in culture medium deprived of IL-3 and seeded in 96-well black clear bottom microplates coated with Poly-D-Lysine (Becton Dickinson Biosciences, San Jose, California, USA) at a density of 20,000 cells per well using Titan Multidrop 384 automatic dispenser (Titertek Instruments, Inc., Huntsville, AL).
Cells were plated onto tissue culture plastic (Falcon, Franklin Lakes, NJ, USA) at a density of ~2.5 × 104 cells/cm.
Cells were seeded on Permanox chamber slides (Nalge Nunc, Naperville, IL, USA) at a density of 550 cells/mm.
Briefly, cells were seeded on a MatTek Petri dish (MatTek, Ashland MA USA) at a density of ∼1.5 × 10 cells per dish.
Cells were cultured in a 96-well cell culture cluster (COSTAR NY, USA) at a density of 4 × 10 cells ml−1.
