represent earlier and later time intervals after seeding, respectively, and represent the number of cells at these intervals. the presence and spatial arrangement of the following molecules in MG-63 cells were evaluated: talin, an integrin-associated protein present in focal adhesion plaques, also reported as a protein of the cell membrane cytoskeleton and known to participate in cell-substrate adhesion; vinculin, another protein of focal adhesion plaques, participating in cell-substrate adhesion Punicalagin inhibitor and stabilizing the focal adhesions;43 beta-actin, an important component of the cell cytoplasmic cytoskeleton, associated with focal adhesion plaques. For immunofluorescence staining of these molecules, the cells on day 3 after seeding were rinsed twice in phosphate-buffered saline and fixed with 70% ethanol (?20C, 20 minutes) pretreated with 1% bovine serum albumin in phosphate-buffered saline containing 0.05% Triton X-100 (Sigma) for 20 minutes at room temperature in order to block nonspecific binding sites and to permeabilize the cell membrane. The cells were then incubated with the following primary mouse monoclonal antibodies: anti-chicken talin (Sigma, clone 8D4) anti-human vinculin (Sigma) and antisynthetic N-terminal peptide of -actin (Sigma). All antibodies were diluted in phosphate-buffered saline to concentrations of 1 1:200 and applied overnight at 4C. After rinsing with phosphate-buffered saline, the secondary antibody, represented by goat anti-mouse F(ab)2 fragment of IgG conjugated with Alexa Fluor? 488 (dilution 1:1000; Molecular Probes) was added for 1 hour at room temperature Punicalagin inhibitor and incubated in the dark. The cells were then rinsed in phosphate-buffered saline, mounted under microscopic glass coverslips in a Gel/Mount permanent fluorescence-preserving aqueous mounting medium (Biomeda Corporation, Foster City, CA) and evaluated under a Leica confocal laser Punicalagin inhibitor scanning microscope (TCS SP2, Germany). Potential immune activation of cells on nanofibrous membranes The secretion of TNF- into the cell culture medium and the concentration of ICAM-1 in cell homogenates were measured in human osteoblast-like MG-63 cells and murine macrophage-like RAW 264.7 cells (American Type Culture Collection, Manassas, VA). The cells were seeded on PLGA or PLGA-ND membranes fixed in CellCrown inserts in polystyrene 24-well cell culture plates (TPP; well diameter 1.5 cm). Each well contained 30,000 cells and 1.5 mL of the culture medium. MG-63 cells were cultured in the Dulbeccos Modified Eagle Minimum Essential Medium mentioned above, and for RAW 264.7, RPMI-1640 medium (Sigma; 10% fetal bovine serum, 40 g/mL gentamicin) was used. As reference samples, pure polystyrene wells and wells with CellCrown inserts were used. For comparison, PLGA meshes with hydroxyapatite (HAp) nanoparticles (Sigma; 200 nm particle size) were used. The concentration of HAp in PLGA was similar as in the case of ND (ie, approximately 23 wt%). After 24 hours, the membranes were transferred into fresh wells filled with cell culture medium in order to exclude the cells adhering on the underlying well bottom from the evaluation. In the reference wells with and without CellCrown inserts, the culture medium was also changed. The cells were then cultivated for an additional 6 days. For each material and cell type, four samples were used. After 6 days of cultivation, the cell culture medium was collected and used for measuring the concentration of TNF- by a sandwich enzyme-linked immunosorbent assay (ELISA) using commercially available kits. A human TNF- Quantikine ELISA kit and mouse TNF- Quantikine ELISA kit were used for the MG-63 and RAW 264.7 cells, respectively. Both kits were purchased from R and D Systems (Minneapolis, MN) and used according to the manufacturers protocol. As a positive control for TNF- production, MG-63 or RAW 264.7 cells grown in polystyrene culture wells and stimulated with lipopolysaccharide (LPS) from (0111:B4, -irradiated, BioXtra, Sigma) were used. Lipopolysaccharide was applied in concentrations ranging from 0 to 10 g/mL on day 6 after seeding for Rabbit polyclonal to ADD1.ADD2 a cytoskeletal protein that promotes the assembly of the spectrin-actin network.Adducin is a heterodimeric protein that consists of related subunits. 24 hours. For each concentration, four wells were used. As a negative control, fresh Dulbeccos Modified Eagle Minimum Essential Medium or RPMI-1640 media supplemented with fetal bovine serum and gentamicin, which were not really subjected to cells, had been utilized. The concentrations of TNF- in cell lifestyle media had been Punicalagin inhibitor driven from calibration curves and provided in pg per 100,000 cells, ie, not really per mL from the lifestyle media, as normal.44,45 The nice factor was that the cell numbers over the tested materials had been different. For MG-63 cells, it ranged typically from 305,600 to 753,900 cells/test, and for Organic 264.7 cells, from 748,900 to at least one 1,220,600 cells/test (Amount 9A and B). Open up in another window Amount 9 Markers of cell immune system activation in individual osteoblast-like MG-63 cells (ACI) and murine macrophage-like Organic 264.7.