Invasion assays

Experiments in cell-free in vitro assays have shown that formation of a PNA triplex invasion complex with a homopurine motif on the template strand results in the steric hindrance of RNA polymerases, leading to complete arrest of further... 

Thompson, S. and Smith, M.T. (1985). Measurement of the diene conjugated from of linoleic acid in plasma by high performance liquid chromatography. A questionable non-invasive assay of free radical activity. Chem. Biol. Interactions 55, 357-366. 

At Leica Biosystems Newcastle Ltd., invasive breast cancer tissue controls, demonstrating HER2 expression levels at 3+, 2+, 1+, and 0, are incorporated into all Oracle HER2 Bond IHC System cell line quality control runs. This ensures that control cell lines are validated as a viable assay control. The evaluation of control cell lines should always be performed within the context of appropriate tolerance limits. Subtle changes from batch to batch may occur, and it is the correct evaluation of the cell line staining patterns within appropriate tolerance limits that enables control cell lines to be utilized both in a commercial setting and as an EQA monitoring device. 

R. Pandian, J.L. Lu, and J. Ossolinska-Plewnia, Fully automated chemiluminometric assay for hyper-glycosylated human chorionic gonadotropin (invasive trophoblast antigen). Clin. Chem. 49, 808-810 (2003). 

In summary, chemiluminescence is a sensitive, non-invasive technique that can measure reactive oxidant production by small numbers of neutrophils indeed, neutrophil-derived chemiluminescence can be detected in as little as 5 fA of unfractionated human blood. The assay is suitable for automation using either multichannel luminometers or luminescence microtitre plate readers. Many researchers, however, have questioned the usefulness of this technique because of the uncertainty of the nature of the oxidant(s) that are detected. Nevertheless, in view of the recent developments made towards the identification of the oxidants measured and the assay s ability to detect intracellular oxidant production, it is has an important place in the phagocyte research laboratory. 

Disulfoton induced the liver MFO system in animals (Stevens et al. 1973). In the same study, exposure to disulfoton orally for 3 days also increased ethylmorphine N-demethylase and NADPH oxidase activities, but had no effect on NADPH cytochrome c reductase. Thus, the induction of the MFO system required repeated dosing with relatively high doses. Furthermore, these changes are not specific for disulfoton exposure, and these subtle liver effects require invasive techniques in humans to obtain liver tissue for performance of these enzyme assays. 

The in vitro invasive assay consisted of grafting rat tracheas by implanting into severe combined immxmodeficiency disease (SCID) mice. The tracheas were first injected with the adenocarcinoma cells to promote tumor growth. Invasiveness was determined by histochemical staining of the iso-... 

Selected entries from Methods in Enzymology [vol, page(s)] Chelation, 238, 74, 76, 297 buffers [for analysis of exocytosis, 221, 132 preparation, 219, 186 modulation of cytosolic buffering capacity with quin2, 221, 159] fluorescence assay, 240, 724-725, 740-742 fluorescence imaging, 225, 531 238, 303-304, 322-325, 334-335 free intracellular levels after bacterial invasion, 236, 482-489 free calcium in solutions for membrane fusion analysis, calculation and control, 221, 149 homeostasis mechanisms, 238, 80 hormonal elevation, 238, 79 inositol phosphate effect on release, 238, 207 determination of cytosolic levels [computer methods, 238, 73-75 with fura-2, 238, 73, 146 with indo-1, 238, 298, 316-317 with quin-2, 238, 297] hormone effects, 238, 79 ionomycin effects, 238, 79 membrane depolarization effects,... 

Two-Dimensional vs. Three-Dimensional In Vitro lUmor Migration and Invasion Assays... 

The aim of this chapter is to focus on two key attributes of malignant cells the ability to move and to spread across tissue boundaries as essential components of invasion and metastasis. It includes a summary of the most accessible, simple, reductionist (2-D) assays which are appropriate for the higher throughput early stage smdies, but also makes the case for the inclusion of more complex (3-D) assays and a description of those that can best be adapted for target validation and drug evaluation. 

Although much has been learned ftom in vitro assays, we do not yet fully understand the predominant migratory mechanisms used by cancer cells in vivo. It is important that any molecular mediators (or their inhibitors) identified in one assay are tested in complementary assays and validated in appropriate in vivo models before they can be assumed to play a significant role in invasion and metastasis. There are several examples where a molecule can have either positive or negative regulatory roles in key cellular functions depending on the cellular/microenvironmental context (e.g., tissue inhibitors of matrix metalloproteinases TIMPs (12)). Thus, care needs to be taken to avoid undesirable activities or, as in the example of some angiogenic inhibitors, compensatory mechanisms that result in adverse events (13). 

Invasion Assays From 2-D Towards 3-D Functional Assays... 

Variations on the filter-based assay have been designed to approximate more physiological contexts. Such assays include tumor cell invasion across a confluent cell monolayer (e.g., endothelial cells (EC) as a surrogate for intravasation or extravasation during hematogenous metastasis (24)) and ovarian carcinoma invasion of mesothelial cell monolayers (25). Additionally, 1 mm thick slices of human brain tissue have been used as a tissue barrier on Transwell filters with invasion of GFP-labeled glioma cells measured by confocal microscopy (26). 

Other assays are being explored in HTS platforms to identify potential inhibitors of invasion, for example, compounds that affect the shape or morphology of cells or their ability to generate invadopodia. Quintavalle et al. recently described such a method initially using Src-transformed NIH 3T3 fibroblasts grown on 384-well optical plates. Imaging of cellular and nuclear morphology combined with phalloidin-stained F-actin was used to discriminate compounds which reduced (or enhanced) the number of... 

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