In addition, the impact of regimen simplification on immunologic parameters was investigated

hild with Lesch-Nyhan, GM02226) and Niemann-Pick type A dermal fibroblasts (NPD, GM13205) were obtained from the Coriell Cell Repository (Camden, NJ), which is part of NIH cell repository and provides scientists with resources for cell and genetic research. These two cell lines have been used in the previous publication[24]. Cells were maintained in high-glucose Dulbecco Minimum Essential Medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Invitrogen). In vitro ASMase Activity Assay In vitro ASMase enzymatic assays were performed as described previously with modifications [25]. Briefly, various tissues were collected from C57/BL6 mice and minced with scissors in lysis buffer (0.2% Triton X-100, 50 mM Tris-HCl, pH 7.4, protease inhibitor cocktail (Roche, Germany)). The tissues were then sonicated briefly, and tissue debris and unbroken cells were pelleted and removed by centrifugation at 800 x g for 5 min at 4�C. Each reaction mixture contains 1 mM EDTA, 250 mM sodium acetate, 100 mM choline-methyl-14C sphingomyelin (Lipidomics Core Facility, at the Medical University of South Carolina, Charleston, SC), and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666200 0.2% Triton X-100, pH 5.0. After incubation for 0.5 h at 37�C, the reaction was stopped by adding 1.5 mL of chloroform/methanol (2:1; Sigma Aldrich, St. Louis, MO), followed by adding 400 L of water. Phases were separated by centrifugation at 2000 x g for 5 min. The upper phase was subjected to scintillation counting. ASMase activity Butein chemical information 19667219″ title=View Abstract(s)”>PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667219 was determined by quantification of the amount of the released radioactive phosphocholine. Electroretinogram (ERG) Recordings The ERG recordings were performed as described previously [26]. Briefly, overnight darkadapted mice were anesthetized using xylazine (20 mg/kg, i.p.)