Rat Amnion Type IV Collagen Composition and Metabolism: MATERIALS AND METHODS(3)

4 May
2013

MATERIALS AND METHODS(3)

This concentration of recombinant adenovirus was selected on the basis of previous experience with transduction of cultured cells and experiments in which rat amnions were infected with lower concentrations of virus. In some experiments, the MMP inhibitor batimas-tat (BB-94: 4-[Af-hydroxylamino]-2R-isobutyl-3S-[thienyl-thiomethyl]-succinyl-L-phenylalanine-A/-methylamide; British Biotech, Oxford, UK) was added to the culture medium at a concentration of 3 ^M in dimethylsulfoxide (DMSO). Control cultures received the DMSO vehicle. Thirty hours after infection, the conditioned media were collected and centrifuged before zymographic analysis for MMP activity and hydroxyproline assay as modified from our previously reported method. birth control yasmin

For the hydroxyproline assay, 50 ^l of the conditioned medium was dried in plastic tubes in an oven at 100°C. Fifty microliters of 4 N NaOH was added, and the tubes were then placed into boiling water for 90 min. Fifty microliters of 1.4 N citric acid was then added to bring the pH to 6.0. One milliliter of chloramine-T solution was then added, and the tubes were incubated at room temperature for 20 min. One milliliter of aldehyde/perchloric acid solution was then added, and the tubes were incubated at 65°C. After 15 min, absorbance at 550 nm was determined. The absorbance values of the starting culture medium were subtracted from those of the conditioned media. Samples were assayed in triplicate.

The amnion tissue was processed for detection of p-ga-lactosidase activity and fixed in formalin for histological examination and analysis of nuclear DNA fragmentation in apoptotic cells.

The recombinant adenoviruses used in these studies were free of wild-type adenovirus on the basis of titration studies carried out in nonpermissive HeLa cells and the absence of E1a in infected cells as assessed by immunofluorescence using an anti-E1a antibody.

Histological Quantitation of Apoptosis

The method of Wijsman et al. was employed to detect nuclear DNA fragmentation in formalin-fixed, paraffin-embedded tissue sections. This immunohistochemical procedure identifies nuclear 3′-end labeled DNA fragments. We have previously reported that apoptosis detected by this method is directly correlated with intranucleosomal DNA cleavage determined by formation of 180- to 200-basepair (bp) DNA ladders. The percentage of apoptotic cells in each specimen was assessed by determining the percentage of dark brown-stained nuclei in 10 random fields viewed at X400, amounting to the scoring of approximately 1000 nuclei/specimen.

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