edius wedding title project free download

                                                  edius wedding title project free download 

edius wedding data

Reagents

Fetal bovine serum (FBS), human transferrin, sodium selenite, lipopolysaccharide (LPS), mouse recombinant interleukin (IL)1β, and nuclear factor kappa B (NF-kB) inhibitor pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma-Aldrich (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium, trypsin-ethylenediaminetetraacetic acid, l-glutamine, and penicillin-streptomycin mixture were obtained from Lonza Group (Basel, Switzerland). Pronase and collagenase P were obtained from Roche Applied Science (Penzberg, Germany).

Recombinant mCRP produced in Escherichia coli had both cysteines replaced with alanine residues and was solubilized by an acylation technique (C-rmCRP) in 25 mM NaPBS (pH7.4), purified and tested in several bioassays to confirm its equivalence to biological mCRP from the pentameric form and free of contaminating endotoxin. The final endotoxin level of all protein solutions was below the detection limit 0.06 EU/ml of the assay [22].

Cell culture conditions and treatments

The murine chondrogenic cell line ATDC5 (RIKEN Cell Bank, Tsukuba, Japan) was cultured in DMEM/Ham’s F12 supplemented with 5% FBS, 10 μg/ml human transferrin, 3 × 10 M sodium selenite, 4 mM l-glutamine, 50 units/ml penicillin and 50 µg/ml streptomycin. Articular cartilage samples were obtained from knee and hip joints of patients undergoing total joint replacement, under written patient consent, and permission from the local ethics committee, according to the declaration of Helsinki. Human primary chondrocytes were isolated and cultured as previously described [23].

Chondrocytes were seeded in six-well plates (2.5 × 105 cells/well) and treated with 10, 25, or 50 µg/ml mCRP, 100 ng/ml LPS or 0.1 ng/ml IL-1β for 24 h, after serum starvation of human primary chondrocytes or ATDC5 cell line for 4 h or overnight, respectively. All the concentrations were selected based on previous studies and in the absence of chondrocyte cell toxicity. All treatments lasted 24 h, after 4 h of starvation for FBS in human primary chondrocytes and overnight starvation in ATDC5 cell line.