2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Virus infection HPIV2 was obtained from the Helsinki University Central Hospital laboratory where it was used as a reference virus. The virus was grown in the GMK cells according to standard procedures . Medium was removed when the adherent GMK, HSG or HSY target cells covered 70–90% of the surface in 75 cm2 cell culture flasks. They were washed with 1 ml culture medium twice and then exposed to 1.5 μg/ml
trypsin at +37°C and in 5% CO2 for 5 minutes. The detached GMK, HSG, BIX 1294 concentration HSY cells were divided into the 6-well plates, which 1.2 × 106 cells per well. 23 μl HPIV2 primary viral suspension, containing 2.7 × 107 infective units per milliliter, was added to each 2.5 ml cell culture well. The cells were harvested at zero hours
(before addition of HPIV2), at two hours, on day one and on day three. In parallel, cells cultured without HPIV2 were harvested as controls at zero and two hours and on one and three days. Double-immunofluorescence staining GMK cells cultured on coverslips for 2 hours, 1 day or 3 days were washed in 10 mM phosphate buffered, 150 mM saline, pH 7.4 (PBS), fixed in pure acetone for 20 minutes at -20°C and incubated in 1) a mixture of 2 μg/ml click here polyclonal rabbit anti-human ADAM9 IgG (Triple Point Biologics, Forest Grove, OR) and fluorescein isothiocyanate (FITC) labeled monoclonal mouse anti-HPIV2 hemagglunin-neuraminidase Oxaprozin IgG1 (Light Diagnostic Respiratory DFA Viral Screening & Identification Kit, Millipore, Temecula, CA, USA) for 30 minutes and 2) Alexa Fluor 594-labeled goat anti-rabbit IgG (Molecular Probes, Eugene, OR) for 30 minutes. Non-immune rabbit IgG and monoclonal mouse IgG1 of irrelevant specificity were used at the same concentration instead of the primary antibodies as negative controls. Synovial membrane-like interface tissue samples collected from the proximal bone-cement or bone-stem interfaces around aseptically loosened femoral stems during revision
total hip replacement operations were used as positive controls . Coverslips were mounted using Vectashield Mounting Medium containing 4′, 6-diamidino-2-phenylindole (DAPI, Vector Laboratories, H 89 concentration Burlingame, CA) for nuclear staining. HSY cells cultured on coverslips for 2 hours, one day or three days were washed in PBS, fixed in pure acetone for 20 minutes at -20°C and incubated in 1) a mixture of affinity purified rabbit anti-human (carboxy-terminal end of, proprietary information) ADAM8 IgG (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) and FITC-labeled monoclonal mouse anti-HPIV2 IgG1 (Millipore) for 30 minutes and 2) Alexa Fluor 594-labeled goat anti-rabbit IgG (Molecular Probes) for 30 minutes. The antibody used for ADAM8 staining has been peptide-affinity purified and does not react with other ADAMs. Coverslips were mounted using Vectashield Mounting Medium containing DAPI (Vector Laboratories).