Quantitation of activated caspase-3 levels in treated microglial cells, by ELISA and immunofluorescence.

<p>(<b>A</b>) After treatment of the primary microglial cell cultures and incubation for a total of 24 hrs as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079160#pone-0079160-g001" target="_blank">Figure 1</a>, cells were lysed and tested by a specific ELISA assay for cleaved caspase-3 levels quantitation. The results shown (ng/mL cleaved casp-3 per µg of total protein) correspond to the mean of four independent experiments (N=4), each one performed with duplicate samples. Bars correspond to SEM. A discontinuous line represents the mean value obtained for untreated cells. Statistically significant differences were calculated by applying the Student’s t test in relation to the values obtained with the corresponding TLR ligand in the absence of αSyn-preconditioning (* <i>p</i><0.05) and with Wt aSyn alone (# <i>p</i><0.05). Treatment with staurosporine from <i>Streptomyces</i> sp. (5 µM) for 6 hrs was used as a positive control. (<b>B</b>) Cells were cultured in appropriate culture plates and treated as explained above (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079160#pone-0079160-g001" target="_blank">Figure 1</a>) for subsequent labelling of cleaved caspase-3 and nuclear Hoechst 33342 staining for IF analysis, as described in the Methods Section. Samples were analyzed under the fluorescence microscope and three images from random fields containing ca. 80-90 cells each, were recorded, and analyzed for fluorescence quantification. The total specific red fluorescence (RF) and blue fluoresce (BF) were measured and the RF/BF ratio was used as a quantitation method and is represented in this figure. The results shown (RF/BF ratio) correspond to the mean of three images analysed (N=3) within one representative experiment, and bars correspond to SEM. A discontinuous line represents the mean value obtained for images from untreated cells. Statistically significant differences were calculated by applying the Student’s t test in relation to the values obtained with the corresponding TLR ligand in the absence of αSyn-preconditioning (* <i>p</i><0.05) and with Wt αSyn alone (# <i>p</i><0.05). Treatment with staurosporine from <i>Streptomyces</i> sp. (5 µM) for 6 hrs was used as a positive control.