The luminescent signal was detected using a compatible CCD imaging and analysis system measuring absorbance at 450 nm. The concentration of each sample was quantified by comparing the spot intensities with the corresponding standard curves calculated from the standard sample results using the SearchLight Array Analyst Software. Integrated density values were proportional FK228 concentration to the concentrations of bound proteins. Standard curves, raw data and final pg/ml concentrations for each analyte and each sample were reviewed in the array software and exported to Microsoft Excel Software for

further statistical analysis. Sample values were calculated from the standard curve in a linear range. All counts were based on individual sections and show the total number of neurons per slice. The number of microscopically detectable immunoreactive ChAT-positive neurons was counted in each whole slice and visualized under the 40 × objective by a blind observer. Multistatistical selleck screening library analysis (KaleidaGraph) was

obtained by one-way ANOVA with Fisher LSD post hoc test, comparing controls against respective treatments in which p < 0.05 represents statistical significance. We were interested in identifying the most efficient transfection method for generating NGF-secreting primary rat monocytes. Each system was Mannose-binding protein-associated serine protease optimized and evaluated for reproducibility and functional

gene expression (NGF secretion). Unfortunately, no NGF secretion was observed in primary monocytes transfected by electroporation, Effectene or FuGene (even following extensive optimization) (Table 1). Note that Table 1 only displays NGF secretion under optimized conditions. Refer to Methods section for all transfection conditions tested. Although the transfection conditions tested within each method were not always equivalent (i.e. DNA concentration) to other methods tested, this was not the reason for different efficiencies between systems. DNA input was determined in accordance with the recommended method levels and thus different concentrations were needed to optimize each method. When primary rat monocytes were transfected using nucleofection, monocytes secreted 0.8 ± 0.2 ng/ml NGF per 24 h per 1 million cells under optimized conditions (determined after many attempts at varying transfection conditions, see Table 1). Approximately 10–30% of nucleofected monocytes were transfected with the pmaxGFP vector (data not shown). However, monocyte nucleofection reproducibility was low (21%). Cell viability was also relatively low in nucleofected cells, where approximately 89% were annexin-V-positive and approximately 51% PI-positive (Fig. 1D–F). Although many attempts were made to enhance reproducibility and determine the factors responsible (i.e.