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Study This And Learn Ways To Get Better At PKC412 Very Easily

In neuron�Cmicroglia mixed cultures, microglia were counter-stained with a primary antibody raised against Iba-1 (goat, 1?:?500) followed by a secondary Alexa Fluor 594 chicken anti-goat antibody (1?:?200) and the nuclei stained with Hoechst 33258 dye. Fluorescence was visualized using a Leica DFC490 camera (Leica Microsystems GmbH, Wetzlar, Germany) adapted to an Axioskope? microscope (Carl Zeiss MicroImaging GmbH, G?ttingen, Germany). Green- and red-fluorescence images of 10 random microscopic fields (original magnification: 400��) were acquired per sample. Evaluation of neurite extension and number of nodes from individual neurons, were traced manually using ImageJ software (National Institutes of Health). Results of at least three different experiments, Obeticholic Acid mw carried out in duplicate, were expressed as mean?��?SEM. Differences between groups were determined by one-way anova with Dunnett��s or Bonferroni��s multiple comparisons post-tests, using Instat 3.05 (GraphPad Software, San Diego, CA, USA). Statistical significance was considered for a p-value <?0.05. Microglial cells are important sources of cytokines in the brain (Hanisch 2002). In fact, a recent report from our group demonstrated that microglia, upon UCB stimulation, engages an inflammatory reaction which entails IL-1��, IL-6 and TNF-�� production and follows different temporal profiles of secretion (Silva et?al. 2010). However, the influence of neighboring cells must be considered when evaluating the response of microglia to toxic molecules like UCB. Indeed, several authors have described that neurons or astrocytes can modulate <a href="http://en.wikipedia.org/wiki/PLEKHO1">PLEKHO1 the inflammatory reaction undertaken by microglia (Giulian et?al. 1993; Mott et?al. 2004; Min et?al. 2006; Polazzi and Contestabile 2006). Hence, we compared cytokine secretion by microglia directly exposed to UCB or to astrocytes- and neuron-conditioned media (ACM and NCM, respectively) after incubation with UCB. Our results indicated that both UCB-treated ACM and NCM completely counteracted (p?<?0.01) the UCB-induced increase in IL-1�� (1.5-fold, p?<?0.01; corresponding to a shift from 1200?pg/mL for control conditions to 1850?pg/mL for UCB), as depicted in Fig.?1(a). Regarding TNF-�� production (Fig.?1b), only <a href="http://www.selleckchem.com/products/s-gsk1349572.html">selleckchem NCM was able to significantly prevent (p?<?0.05) the elevation elicited by UCB (1.5-fold, p?<?0.05; corresponding to a shift from 970?pg/mL for control conditions to 1400?pg/mL for UCB). Curiously, both ACM and NCM obtained after treatment with UCB led to an IL-6 overproduction by microglia (2.7- and 2.8-fold, respectively; p?<?0.01 vs. control; corresponding to approximately 4800?pg/mL for both conditions) which proved to be significantly higher (p?<?0.01 for both UCB-treated ACM and NCM) than the one triggered by the direct effect of UCB in microglia (ranging from 2060?pg/mL for control conditions to 2400?pg/mL for UCB, as can be seen in Fig.?1(c).</div>
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