Normal mouse IgG was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary mouse cortical neuron culture and experimental conditions Mice were housed in community cages under a 12 h light/dark cycle at 20C22C and fed 2004). together, these data suggest that parallel and redundant pathways of oxidative stress and caspase-mediated cell death are involved. We conclude that CD47 mediates neuronal cell death through caspase-dependent and caspase-independent pathways. 2004), and leukemia cells (Mateo 1999, 2002; Saumet 2005; Bras 2007). Because many fundamental mechanisms of cell death are known to be highly conserved between multiple cell types, it is possible that CD47 may possess neurotoxic actions as well. CD47 is present in neuronal cells, 2-Methoxyestradiol and one study showed that viral over-expression of CD47 in neuron induced apoptosis (Koshimizu 2002). In this study, we used primary mouse cortical neurons to investigate the mechanisms of CD47-induced neuronal death. Specifically, we asked whether ligand-mediated activation of CD47 is neurotoxic, and if so, whether downstream pathways of oxidative stress and caspases are involved. Materials and methods Reagents Neurobasal media, B27 supplement, 0.05% trypsinCEDTA, L-glutamine, antibiotics, and fetal bovine serum for cell culture were from Gibco (Rockville, MD, USA). TSP and U83836E were purchased from Calbiochem (San Diego, CA, USA). 4N1K (KRFYVVMWKK) was from Sigma Genosys (The Woodlands, TX, USA) and was dissolved in sterile ddH2O at a concentration of 100 mg/mL as a stock solution. This stock was aliquoted and stored at ?80C. 5-(and-6)-Chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) was purchased from Molecular Probes (Eugene, OR, USA). Caspase inhibitors (z-VAD-fmk and z-DEVD-fmk) were from R&D (Minneapolis, MN, USA), and were dissolved in dimethyl sulfoxide at a concentration of 20 mM as a 2-Methoxyestradiol stock solution. Rabbit anti-caspase 3 primary antibody was purchased from Cell signaling (Danvers, MA, USA). Anti-mouse CD47 monoclonal antibody (Clone miap301) was from BD Pharmingen (San Jose, CA, USA). Normal mouse IgG was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary mouse cortical neuron culture and experimental conditions Mice were housed in community cages under a 12 h light/dark cycle at 20C22C and fed 2004). In the inhibitors experiment, cultures were pre-treated with inhibitors for 1 h prior to addition of 100 g/mL 4N1K for 24 h. Neurotoxicity was evaluated by the standard lactate dehydrogenase (LDH) release assay (Roche Diagnostics, Mannheim, Germany) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St Louis, MO, USA). All LDH and MTT assays were repeated at least three times in triplicate. Reactive oxygen species measurement Levels of cellular reactive oxygen species (ROS) were measured using CM-H2DCFDA. Briefly, after the neurons were treated with 10 g/mL TSP or 100 g/mL 4N1K for various times (0, 3, 6, 12, and 24 h), CM-H2DCFDA was added to the neuron cultures to a final concentration of 1 1.25 M, and incubated for 30 min at 37C. The amount of intracellular oxidants is proportional to the intensity of fluorescence. The fluorescence intensity of the cells was used as an indicator of the production of ROS, and analyzed by flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA). Western blotting analysis Caspase 3 activation was determined by western blot. Cells were lysed in cell lysis Rabbit Polyclonal to SRPK3 buffer (Cell signaling) in the presence of protease inhibitors. Insoluble materials were removed 2-Methoxyestradiol by centrifugation (20 800 tests (SPSS version 11.5, SPSS Inc., Chicago, IL, USA). Statistical significance was at 0.05. Results Activation of CD47 is neurotoxic Neurotoxic effects of CD47 were evaluated using a standard LDH release assay. Exposure to the CD47 ligand TSP (0.5C10 g/mL) for 24 h induced a dose-dependent cell death in primary cortical mouse neurons (Fig. 1a). Pre-treatment with a CD47 blocking antibody for 1 h significantly decreased TSP-induced neuronal death (Fig. 1b). The specificity of this pathway was further confirmed using 4N1K, a CD47-specific activating peptide. Exposure to 4N1K (12.5C100 g/mL) induced a similar neurotoxic response (Fig. 1c). To further confirm these LDH neurotoxicity findings, we 2-Methoxyestradiol also measured cell viability using an MTT technique. Neuronal cell viability.
Normal mouse IgG was from Santa Cruz Biotechnology (Santa Cruz, CA, USA)
Previous article(A,B) Period classes of ICP and CPP adjustments after induction of SAH or sham medical proceduresNext article Next, we assessed the signs of intoxication through measurement of cell trans-epithelial electrical resistance (TEER) and cell viability, colon epithelium permeability to macromolecules, gene expression of TJ proteins, as well as the immunofluorescence (IF) analysis of the cytoskeletal structure and TJ proteins in IPEC-J2 cells