Numerical information symbolize imply six SD. p,.05 vs. CON, + p,.05 vs. corresponding CM group(hereinafter referred to as conditioned medium, CM) was then harvested and centrifuged (300 rpm, 3 min, to eliminate residual mobile material) supernatants were then both concentrated or extracted to exclude DEX. For focus, supernatants had been run by means of Vivaspin columns (Vivaspin20, Sartorius, Aubagne, France) to focus peptides with an Mr .three kD smaller sized molecules, including DEX at an initial concentration of 1025 M, had been washed out serial dilution-concentration steps to attain a approximated final concentration of DEX that was ,3.10211 M. To extract DEX, supernatants were run through Speedisk H2OPhobic DVB polymer columns (JT Baker, Phillipsburg, NJ). Full removal of DEX from CM and DCM was evidenced by the disappearance of the phobic indicator, 5-Pyrimidinecarboxamide,N-hydroxy-2-[methyl[[2-[6-(methylamino)-3-pyridinyl]-4-(4-morpholinyl)thieno[3,2-d]pyrimidin-6-yl]methyl]amino]- phenol purple.Whole RNA was isolated (RNAeasy kit Qiagen, Hilden, Germany) and reverse transcribed with Superscript II RNA Hreverse transcriptase (Invitrogen) and custom-synthesized OligodT12-eighteen primers (MWG Biotech, Ebersberg, Germany). Quantitative PCR (qPCR) was carried out with a LightCycler (Roche, Mannheim, Germany) in 10 ml mixtures that contains two ml of 5X master mix (FastStart DNA SYBR green I Roche), 5 ml of drinking water,.five ml of every single primer and two ml of extracted DNA. The response was carried out with preliminary denaturation for ten min at 95uC (slope, 20uC/s), followed by forty cycles of denaturation at 94uC (5 s), annealing (5 s) at 65uC and extension at 72uC (10 s). Relative mRNA expression ratios (housekeeping genes: actin and gapdh) had been subsequently calculated.Fragmentation of higher molecular fat (HMW) DNA was assayed by pulse-discipline gel electrophoresis (CHEF-DR II Biorad, Hercules, CA). About five.106 astrocytes for every condition had been suspended in 40 ml of PBS, blended with an equivalent quantity of warm 1% Seakem gold agarose (SKG Bio Whittaker Molecular Apps, Rockland, MD) in .5X TBE buffer (forty five mM Tris, forty five mM boric acid, one mM EDTA, pH 8.3), and transferred to block molds (Biorad). Agarose blocks were incubated at 50uC in one ml of NDS buffer (one% laurylsarkosyl, ten mM Tris, .five M EDTA, pH nine.5) containing 200 mg/ml proteinase K, and then in NDS buffer made up of 10 mg/ml RNase each incubation lasted 24 h. The blocks had been inserted into wells 21957443of a one% SKG gel in .5X TBE, and electrophoresed 
at 6V/cm for fourteen h at 14uC with a change time of fifty s.
