Rat Amnion Type IV Collagen Composition and Metabolism: MATERIALS AND METHODS(2)

3 May
2013

In Vitro Basement Membrane Degradation Assays

Basement membranes were prepared from rat amnions and maternal kidney as previously described. The basement membrane extracts (1 mg dry weight) were incubated with bacterial collagenase (Worthington, Freehold, NJ; 18.5 U/mg) or active MMP-9 enzyme (Calbiochem, San Diego, CA; 1 ^g of enzyme/mg of detergent-extracted basement membrane) over a 2-h period or 5-h period, respectively, at 37°C. Hydroxyproline in peptides released during the digestion was quantitated as previously reported. buy flovent inhaler

Infection of Rat Amnion Organ Cultures with Recombinant Adenovirus

Amnions were collected on Day 18 of pregnancy, washed free of amniotic fluid in PBS, and placed into organ culture in 1 ml of serum-free Kennett’s HY medium (Gibco BRL Life Technologies, Gaithersburg, MD) containing 1% BSA in 12-well plastic dishes (2 or 3 amnions/well; within each experiment the number of amnions per well was equal in all treatment groups). Organ cultures were incubated at 37°C under an atmosphere of 5% CO2:95% humidified air. The amnions were infected with either recombinant adenovirus expressing human proMMP-9 (AdMMP-9) constructed as previously reported, or with a control recombinant adenovirus containing the LacZ cDNA, at 5.8 X 108 plaque-forming units (pfu)/well immediately after being placed into culture.

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