5% FCS) on 24-well collagen-coated culture plates. Effectene Transfection Reagents were prepared in glia culture medium (without antibiotics/antimycotics) and added drop-wise to the cells. The transfection method was optimized by testing the effects of: the number of cells added, prolonged incubation time, and removal of complexes after 16 h (data not shown). Cells were incubated with transfection complexes

for 24 h. After incubation, cell supernatants and extracts were collected for further use. Primary astrocytes were isolated as previously done (Wiesenhofer and GSK126 cost Humpel, 2000 and Zassler et al., 2005a) and used as a positive control. Primary cultures of freshly isolated rat monocytes were transiently transfected with pEF-NGF plasmid using FuGene HD Transfection Reagent (Promega) according to manufacturer’s instructions.

Briefly, cells were seeded 1 × 105 cells per well in medium (without antibiotic/antimycotics). Cells were incubated at 37 °C until reaching 80% confluency on the day of transfection. On the day of transfection, the DNA-FuGENE mix was prepared in Optimem (Gibco) and added drop-wise to the cells. Different concentrations of DNA, amount of FuGENE BKM120 HD reagent, incubation times with transfection mix, ‘boosting’ with transfection mix, and recovery times were also evaluated (data not shown). Cells were incubated with transfection complexes for 24, 48, or 72 h in Amaxa culture medium (10% FCS, 2 mM glutamine, 1 ng/ml M-CSF, 1 ng/ml GM-CSF). After incubation, cell supernatants were collected for further use. Primary cultures of freshly isolated rat monocytes were nucleofected with pEF-(−), pmaxGFP, or pEF-NGF using the Human Monocyte Nucleofection kit (Amaxa) according to the manufacturer’s instructions. Monocytes were pelleted directly following isolation at 250 ×g for 5 min. Cell pellets

were resuspended in 110 μl of Nucleofector solution (Amaxa), mixed with plasmid DNA, and transferred to an Amaxa cuvette. Nucleofection was performed using the Amaxa program Y-001. Control samples were nucleofected using RVX-208 the empty vector (pEF-(−)). Immediately following nucleofection, 500 μl of pre-warmed glia culture medium (Optimem I, 5% horse serum, 0.5% FCS) (without antibiotics/antimycotics) or Amaxa culture medium (10% FCS, 2 mM glutamine, 1 ng/ml M-CSF, 1 ng/ml GM-CSF) was added to the cuvette and subsequently transferred to a collagen-coated 24-well culture plate. Nucleofected cells were incubated for 1–2 days at 37 °C 5% CO2. After incubation, cell supernatants and extracts were collected for NGF ELISA or cells were stained for further microscopic analysis. Primary astrocytes were isolated as previously done ( Wiesenhofer and Humpel, 2000 and Zassler et al., 2005a) and used as a positive control.