Microtubules (MT) are the most rigid component of the cytoskeleton. Nevertheless, they often appear highly curved in the cellular context and the mechanisms governing their overall shape are poorly understood. Currently, in vitro microtubule analysis relies primarily on electron microscopy for its high resolution and Total Internal Reflection Fluorescence (TIRF) microscopy for its ability to image live fluorescently-labelled microtubules and associated proteins. For three-dimensional analyses of microtubules with micrometer curvatures, we have developed an assay in which MTs are polymerized in vitro from MT seeds adhered to a glass slide in a manner similar to conventional TIRF microscopy protocols. Free fluorescent molecules are removed and the MTs are fixed by perfusion. The MTs can

Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca2+]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca2+ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca2+]i in the range of 0.1-39.8 µM in small cells like those of

Circulation of cerebrospinal fluid (CSF) plays an important role during development. In zebrafish embryo, the flow of CSF has been found to be bidirectional in the central canal of the spinal cord. In order to compare conditions and genetic mutants across each other, we recently automated the quantification of the velocity profile of exogenous fluorescent particles in the CSF. We demonstrated that the beating of motile and tilted cilia localized on the ventral side of the central canal was sufficient to generate locally such bidirectionality. Our approach can easily be extended to characterize CSF flow in various genetic mutants. We provide here a detailed protocol and a user interface program to quantify CSF dynamics. In order to interpret potential changes in CSF flow profiles, we