Material
DMF and MMF (sodium salt) for all experiments were obtained from Biogen Idec, Carl-Zeiss-Ring 6 85737 Ismaning, Germany and solubilized in dimethylsulfoxide (DMSO), which was also used as the vehicle control. The pH of all media was kept constant at 7.4. Cell culture dishes were from Greiner Bio-One, Maybachstraße 2., 72636 Frickenhausen, Germany; DMEM cell culture medium, sterile phosphate buffered saline were from PAA, Unterm Bornrain 2, 35091 Cölbe, Germany; penicillin, streptomycin were from Gibco/Life Technologies, Frankfurter Straße 129B, 64293 Darmstadt, Germany; cryopreserved primary dissociated cortical cultures from embryonic rats were from QBM Cell Science Inc., 1200 Montreal Road, Building M23A, Suite 147, Ottawa, Ontario, Canada; Cell Titer Blue was from Promega, Schildkrötstraße 15 68199 Mannheim, Germany; the high contact imaging microscope, the anti-CD3 antibody, 7-AAD and Annexin V PE were from Becton Dickinson, Tullastr. 8-12, 69126 Heidelberg, Germany; the anti-Nrf2 antibody was from Santa Cruz Biotechnology, Bergheimer Straße 89, 69115 Heidelberg, Germany; the anti-Actin antibody and secondary antibodies were from Millipore, 290 Concord Road Billerica, MA 01821, USA; the anti-NF-κB-antibody was from Cell Signaling Technologies, 3 Trask Lane Danvers, MA 01923, USA; multi-electrode arrays were from Multichannel Systems, Aspenhaustrasse 21. 72770 Reutlingen, Germany; the MEA analyzing software Spanner was from RESULT software, 47918 Tönisvorst, Germany; the Universal Probe LibraryTM was from Roche, Emil-Barell-Str. 1 79639 Grenzach-Wyhlen, Germany; Fam-Tamra labeled oligonucleotides were from Eurofins-MWG-Operon, Anzingerstr. 7a, 85560 Ebersberg, Germany; the TNFα ELISA was from R&D Systems, Borsigstrasse 7. 65205 Wiesbaden, Germany; the Immunospot Analyzer was from CTL, 2860 Fisher Road, Columbus, OH 43204, USA; Prism software was from GraphPad Software, 2236 Avenida de la Playa, La Jolla, CA 92037, USA; spreadsheet software was from Microsoft, Konrad-Zuse-Str. 1, 85716 Unterschleißheim, Germany; all other chemicals were from Sigma Aldrich, Georg-Heyken-Str. 14 D-21147 Hamburg Germany.
Cell culture, viability assays and glutathione measurement
The preparation of embryonic primary cortical cultures and splenocyte cultures from C57BL/6 and SJL mice and the cell culture of HT22 and fibroblast cells were performed as described [
15,
16]. For the analysis of network activity, cryopreserved primary dissociated cortical cultures from embryonic rats (embryonic day 18, E18, QBM Cell Science) were employed. After thawing, the cells were plated at a final density of 10
5 cells on PDL-/laminin-coated multi-electrode arrays (MEAs) or coverslips. Neuronal cultures were incubated in a humidified atmosphere (5% CO
2/95% air) at 37 °C for 24 h in DMF or vehicle prior to glutamate treatment. Viability was quantitated 24 h after glutamate addition by the Cell Titer Blue (CTB) assay (Promega) and normalized to vehicle treatment. Total glutathione was measured enzymatically as described previously [
15] and normalized to cellular protein measured by the bicinchoninic acid-based method (Pierce). Glutathione released into the cell culture medium was also quantitated enzymatically after 4 h in cystine-free medium and normalized to total cellular protein. Cell viability of splenocytes was assessed using flow cytometry quantitating 7-AAD (BD Pharmingen #51-68981E) and Annexin V PE (BD Pharmingen #556421) stained cells according to manufacturers’ protocols.