Quantitative PCR Neurospheres derived from the subventricular zone were stimulated with 10 ng ml recombinant human GM CSF. 3 days after addition of GM CSF, cells were harvested for the RNA preparation, whereas untreated cells served as con trol. RNA of the GM CSF treated and untreated selleckchem neuro spheres of the SVZ was isolated using the Qiagen RNeasy mini kit following the manufacturers recommendations. cDNA was synthesized from 5 g total RNA using oligodT primers, superscript II reverse transcriptase using standard conditions. Inhibitors,Modulators,Libraries Quantitative PCR was performed using the Lightcycler system with SYBR green staining of DNA dou blestrands. Cycling conditions were as follows beta III tubulin 3 min 95 C, 5 sec 95 C, 10 sec 65 C, 30 sec 72 C. 10 sec 87 C for 50 cycles. NSE 3 min 95 C, 5 sec 95 C, 10 sec 58 C, 30 sec 72 C.

10 sec 81 C for 50 cycles. PLP 3 min 95 C, 5 sec 95 C, 10 sec 62 C, 30 sec 72 C. 10 sec 84 C for 50 cycles. GFAP 3 min 95 C, 5 sec 95 C, 10 sec 60 C, 30 sec 72 C. 10 sec 81 C for 50 cycles. Melting curves were determined using the following parameters 95 C cooling to 50 C. ramping to 99 C at 0. Inhibitors,Modulators,Libraries 2 C sec. The following primer pairs were used rat beta III tub 716s Inhibitors,Modulators,Libraries rat beta III tub 1022as The Lightcycler PCR was performed using the SYBR green master mix, following the manufacturers recommendations. Specificity of product was ensured by melting point analysis and agarose gel electrophoresis. cDNA content of samples was normalized to the expression level of Cyclo philin. Relative reg ulation levels were derived after normalization to cyclo philin, and comparison to the untreated cells.

Luciferase assay To generate the pGL3 p III tubulin experimental vector, the class III tubulin gene promoter was inserted into the pGL3 Basic firefly luci ferase reporter vector as described before. Cultivation of stem cells, DNA transfection and luciferase assay were carried out as described before. Briefly, stem Inhibitors,Modulators,Libraries cells were seeded at a density of 35 000 cells well and were cotransfected with the pGL3 p III tubulin vector and a Renilla luciferase construct with the Lipofectamine method Inhibitors,Modulators,Libraries 24 h after plating. Following the incubation of transfected cells for 24 h, cells were stimu lated with various concentrations of GM CSF in Neuroba sal medium for 48 h. As positive control for in vitro differentiation, stem cells were treated by withdrawing mitogens and adding 5% fetal calf serum.

Using the Dual Luciferase Reporter Assay System the ratio of luminescence signals from firefly http://www.selleckchem.com/products/Erlotinib-Hydrochloride.html and renilla luciferase was obtained. FACS analysis For differentiation experiments, adult NSCs were plated in 15 cm2 culture flasks at a density of 4 million cells and were treated once with 10 ng ml GM CSF. A single cell suspension was made by triturating the neurospheres in 1 ml plastic pipettes, and then collected by centrifuga tion. After resuspension in 1�� phosphate buffered saline, the cells were fixed with 1% PFA.