Modulatory Role for Substance P: MATERIALS AND METHODS(1)

MATERIALS AND METHODS(1)Animals and Cell Dispersion Pituitary glands were obtained from prepubertal Large White-Landrace female pigs (5-6 mo old). This animal model was selected because of its commercial availability and to avoid variations associated with the estrous cycle that might arise in mature females. In the slaughterhouse, animals were killed after electrical stunning and immediately decapitated, and their pituitaries were removed and transferred to sterile cold (4°C) culture medium consisting of Eagle’s Minimum Essential Medium with D-valine (MEM; Sigma, London, UK) supplemented with 0.3% BSA (fraction V; Sigma), 0.58% Hepes (Sigma), 0.22% sodium bicarbonate, and antibiotics. Cell dispersion was carried out essentially as described in detail previously with minor modifications. In brief, anterior pituitaries were cut into 1- to 2-mm3 fragments, decanted, rinsed with MEM, and then exposed sequentially to 0.2% trypsin (type I), 0.1% collagenase (type V), 0.1% soybean trypsin inhibitor (type I), 4 (Jig/ml deoxyribonuclease (type I), and Ca2+/ Mg2+-free salt solution containing EDTA (2 mM and 1 mM; all purchased from Sigma). A final step of mechanical dispersion rendered monodispersed pituitary cells.


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