Luperox tbh70x

Skin fibroblasts were generated from 6 AD patients with different allelic combinations of APOE (one also presenting a the G206D mutation in PSEN1) and six age-matched healthy controls (Table 1). The patients and control subjects were recruited and signed informed consent, previously accepted by the Human Research Ethics Committees Ethics of Spanish National Research Council (CSIC) and CIBERNED (Instituto de Salud Carlos III). All samples were sequenced at the laboratory of Dr. Joan Comella at the Hospital Vall d’Hebron (Lonza, Barcelona, Spain), according to the protocol established by Calero et al. (2009) (link). Fibroblasts were maintained in DMEM (Lonza, Barcelona, Spain) with 10% FBS (Life Technologies, Alcobendas, Spain), 1% penicillin-streptomycin (Lonza, Barcelona, Spain), and 0.1% amphotericin B (Invitrogen, Madrid, Spain). In oxidative stress induction experiments, fibroblasts were treated with tert-Butyl hydroperoxide (tBHP, Luperox^® TBH70X, Sigma Aldrich, Madrid, Spain) at different concentrations. Experiments were conducted in all fibroblasts in parallel.

Tert-Butyl hydroperoxide solution (Sigma-Aldrich, Luperox TBH70X, 458139) was purchased. All other chemicals were of reagent grade.

L-Ascorbic acid, reagent grade, crystalline catalogue number A7506-25G, Luperox^® TBH70X, tert-Butyl hydroperoxide solution catalogue number 458139, bisBenzimide H 33342 trihydrochloride catalogue number B2261, and Red Oil-O catalogue number O0625 were purchased from Sigma-Aldrich and used without additional purification. An XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) proliferation kit with catalogue number 20-300-1000 was purchased from Biological Industries.

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay [36 (link)] was used to detect possible cytotoxic effects and effects of VSOPs-labelling on the proliferation of hASCs. Unlabelled hASCs served as negative controls. Human ASCs treated with 0.1 mM tert-butylhydroperoxide (t-BHP; Luperox® TBH70X; Sigma-Aldrich), which induces cell apoptosis [37 (link)], were used as positive controls. For each patient, 8 wells were seeded at each time point and used to calculate mean extinction values. The analyses were performed 24 h after the labelling procedure and after 7, 14, 21 and 28 days. After removal of the medium from each well, 100 µL MTT were added. After an incubation step at 37 °C in a 5% CO₂ atmosphere for 4 h and removal of the MTT solution, 100 µl isopropanol were added. Thirty minutes later, the color conversion at 570 nm was determined with a Titertek Multiscan PLUS (MKII) multiplate reader (Pforzheim, Germany). The mean extinction values were calculated from 8 wells per patient. The values of the unlabelled hASCs were adjusted to a viability of 100% (viability of untreated ASCs). Viability of the labelled hASCs and the positive controls was presented as a percentage of the viability of the unlabelled hASCs.