When the fluorescence signal of a dye is being quantified, the staining protocol is an important factor in ensuring accuracy and reproducibility. Increasingly, lipophilic dyes are being used to quantify cellular lipids in microalgae. However, there is little discussion about the sensitivity of these dyes to staining conditions. To address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification (Nile Red and BODIPY) or a lipophilic dye commonly used to stain neuronal cell membranes (DiO), and fluorescence was measured using flow cytometry. The concentration of the cells being stained was found not to affect the fluorescence. Conversely, the concentration of dye significantly affected the fluorescence intensity from either insufficient saturation of the cellular lipids or formation of dye precipitate. Precipitates of all three dyes were detected as events by flow cytometry and fluoresced at a similar intensity as the chlorophyll in the microalgae. Prevention of precipitate formation is, therefore, critical to ensure accurate fluorescence measurement with these dyes. It was also observed that the presence of organic solvents, such as acetone and dimethyl sulfoxide (DMSO), were not required to increase penetration of the dyes into cells and that the presence of these solvents resulted in increased cellular debris. Thus, staining conditions affected the fluorescence of all three lipophilic dyes, but Nile Red was found to have a stable fluorescence intensity that was unaffected by the broadest range of conditions and could be correlated to cellular lipid content. ' 2012 International Society for Advancement of Cytometry

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NGF is known to regulate the development and survival of select populations of neurons via its binding/activation of the TrkA and p75NTR receptors. However, NGF dysregulation can result in debilitating pathologies. Thus, the identification of small molecules which inhibit NGF signaling have significant therapeutic potential. PD 90780, Ro 08‐2750, and ALE 0540 are small molecules that have been reported to bind and inhibit NGF activity. Importantly, the docking site of these compounds is hypothesized to occur at the loop I/IV cleft of NGF ‐ a region which is required for efficient and selective binding of a neurotrophin to its receptor(s). Molecular modeling predicts these molecules share conserved molecular features and have the ability to bind and modify the molecular topology of NGF. In order to understand the binding mechanism, we synthesized a pyrazoloquinazolinecarboxilate (PQC) analogue series and tested each compound in an NGF‐dependent PC12 cell differentiation assay. In vitro data confirms that the PQC analogues functionally inhibit NGF's agonist effects on PC12 cell differentiation. The results of this study provide evidence to refine the docking mode of PQC‐like compounds for the purposes of inhibiting NGF in vitro. In addition, we identified series analogue PQC 083 which is markedly better than previously described NGF antagonists.