These kits have been optimized with the ArrayScan® HCS Reader using the Morphology BioApplication Software Module. Thus, automated plate-handling, focusing, cell image acquisition/processing, and data analysis/management are combined in one high-content screening (HCS) system to assay for test compounds. In addition to HCS instruments, cells labeled by the kit reagents can be viewed and analyzed by other fluorescence microscopes.
Figure 2. Staining of cytoskeleton structure in NIH3T3 cells (left, 20X objective) and HMVEC-L cells (right, 20X objective). Markers were detected according to the kit protocol. Cell images were acquired using the Cellomcs ArrayScan® HCS Reader. Colors are F-actin (red), tubulin (green), nuclei stained with DAPI (blue).
Background
The intracellular meshwork of the cytoskeleton is responsible for maintaining cell shape, cell movement, cytokinesis and organelle organization. The cytoskeleton network also facilitates proper function of other proteins by direct binding, transporting, repositioning and sequestering these proteins. The structure of cytoskeleton is controlled by cytoskeleton-associated proteins in response to the external signaling. Therefore, defects in the ability to regulate the dynamics of cytoskeletal structure are likely to cause detrimental effects on other cell function.
Cytoskeletal rearrangement is often associated with cellular toxicity, pathology and cell death. Signaling defects in conjunction with cytoskeletal rearrangement can also contribute to cell proliferation and tumor cell activation, which result in metastasis. An accurate assay for cytoskeletal rearrangement is essential to evaluate potential therapeutic targets.
High-content analysis (HCA) involves a fluorescence cell-based assay in which cells are automatically imaged and analyzed using fluorescence microscopy. The Multiplexed Cytoskeletal Rearrangement HCS Reagent Kits provide a highly effective tool for studying the cytoskeletal changes. The reagents combined with the Cellomics ArrayScan HCS Reader and the Morphology BioApplication Software Module allows accurate quantification of microfilaments and microtubules simultaneously within the same cell.
Figure 3. Staining of DAPI (nucleus, blue), F-actin (microfilament, red) and tubulin (microtubule, green) in NIH 3T3 cells. Cells were treated with 10 μM cytochalasin D or 1 μM staurosporine for 3 hours or incubated only with media (non-treated). Markers were detected according to the kit protocol. Cytochalasin D and staurosporine dismantle cytoskeleton structure, which causes dramatic morphological change in cells. The cell images were acquired using a Cellomics ArrayScan HCS Reader. Graphs are dose response curves for NIH3T3 cell data representing mean ± SD from three plates (sixteen wells per point in a plate) and analyzed using the Morphology BioApplication Software Module.
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