Astrocytes were collected by trypsin digestive function, seeded onto 35- or 100-mm dishes, and used for experiments 6C8 days after replating. Determination of TGF-1 Levels in the Astrocyte Medium Astrocyte-conditioned medium was collected and subjected to acid treatment procedure. in the culture media through the conversion of latent TGF-1 to mature TGF-1. Unlike fluoxetine, both serotonin and sertraline did not stimulate the astrocyte release of active TGF-1. We conclude that fluoxetine is neuroprotective against A toxicity a paracrine signaling mediated by TGF-1, which does not result from a simplistic SERT blockade. with A1-42 oligomers (1 M) for 48 h both in Rabbit Polyclonal to CDKL1 the presence and in the absence of fluoxetine (100 nM C 1 M). Neuronal injury was assessed by the methyltetrazolium test (MTT) assay in LTX-315 pure neuronal cultures, and Trypan Blue staining in mixed neuronal cultures 48 h after A1-42 treatment. For MTT assay cells were incubated LTX-315 with MTT (0.9 mg/ml final concentration, St Louis, MO, USA) for 2 h at 37C. A solubilization solution containing 20% SDS was then added for an additional 1 h and formazan production was evaluated in a plate reader ( = 560 nm). A toxicity in mixed neuronal cultures was assessed by counting dead neurons stained with Trypan blue. Stained neurons were counted in three random microscopic fields/well. Pure Cultures of Cortical Astrocytes Cortical glial cells were prepared from 1- to 3-day-old Sprague-Dawley rats. After removal of meninges and isolation of cortices, cells were dispersed by mechanical and enzymatic dissociation using a 0.25% solution of trypsin (Invitrogen). Cells were plated onto 75-mm2 flasks and maintained in DMEM, supplemented with 10% fetal calf serum, penicillin/streptomycin (100 U/mlC100 g/ml), and glutamine (2 mM). All medium constituents were from Invitrogen, and all plastic materials were from Corning Life Sciences (Lowell, MA, USA). Confluent cultures at 8C10 days were shaken overnight at 37C to remove microglia and oligodendrocytes. Astrocytes were collected by trypsin digestion, seeded onto 35- or 100-mm dishes, and used for experiments 6C8 days after replating. Determination of TGF-1 Levels in the Astrocyte Medium Astrocyte-conditioned medium was collected and subjected to acid treatment procedure. Samples were acidified to a pH of approximately 2.6 with 1 N HCl for 15 min at room temperature, then neutralized to approximately pH 7.6 with 1 N NaOH. Levels of TGF-1 released into the medium were measured by enzyme-linked immunosorbent assay using the TGF1 Emax Immunoassay System (Promega, Madison, WI, USA), based on an antibody sandwich format, strictly following the manufacturers instructions. In LTX-315 brief, 96-well plates were coated overnight at 4C with primary monoclonal anti-TGF-1 antibody. A blocking solution was added for 35 min at 37C before incubation with samples and standards for 90 min at room temperature, to allow binding of soluble TGF-1. A primary polyclonal anti-TGF-1 antibody was then added for 2 h to bind captured TGF-1. Finally, specifically bound polyclonal antibody was detected by incubation for 2 h with a horseradish peroxidase-conjugated secondary antibody. Wells were extensively washed between each step. After a final 10-min incubation with achromogenic substrate solution, the resulting redox reaction was stopped by acidification with 1N HCl, and absorbance was immediately measured at 450 nm. The assay is sensitive in the range of 32C1000 pg/ml. Western Blot Western blot analyses was performed as previously described (Caraci et al., 2015a) on neurons or astrocytes harvested at 4C in RIPA buffer in the presence of a cocktail of protease inhibitors (SigmaCAldrich P2714), serine/threonine phosphatase inhibitors (SigmaCAldrich, P0044) and tyrosine protein phosphatases inhibitors (SigmaCAldrich, P5726). Protein concentrations were determined by Bradfords method using bovine serum albumin as a standard. After blocking, membranes were incubated with the following primary antibodies overnight at 4C: rabbit anti-TGF-1 (Abcam 25121, Cambridge, UK; 1:1000), rabbit anti-MMP2 (Santa Cruz Biotechnology, Santa, CA, USA; 1:500) and mouse anti–Tubulin and anti–Actin (SigmaCAldrich; 1:500). Secondary goat anti-rabbit labeled with IRDye 680 (1:30.000 Li-COR Biosciences) and goat anti-mouse labeled with IRDye 800 (1:25.000 Li-COR Biosciences) were used at RT for 45 min. Hybridization signals were detected with the Odyssey Infrared Imaging System (LI-COR Biosciences). Western blot data were quantified by densitometric analysis of the hybridization signals in four different blots per experiment. Gene Expression Analysis by Real-Time RT-PCR Total RNA was isolated from cultured astrocytes treated with fluoxetine (1 M) using TRIzol reagent (Invitrogen), GenEluteTM Mammalian Total RNA Miniprep Kit and DNASE70-On-Column DNase I Digestion Set (St Louis, MO, USA) as previously.
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