This study demonstrates the importance of the hydrodynamic environment in microfluidic systems in quantitative cellular assays using live cells. Commonly applied flow conditions used in microfluidics were evaluated using the quantitative intracellular Ca 2+ analysis of Chinese hamster ovary (CHO) cells as a model system. Above certain thresholds of shear stress, hydrodynamically induced intracellular Ca 2+ fluxes were observed which mimic the responses induced by chemical stimuli, such as the agonist uridine 5′-triphosphate tris salt (UTP). This effect is of significance given the increasing application of microfluidic devices in high-throughput cellular analysis for biophysical applications and pharmacological screening.Cell-based assays have been performed in a variety of sectors in the life sciences, particularly those associated with biotechnology and the pharmaceutical industries. Many cellular assays use intact cells to obtain functional information on cell signaling pathways as well as kinetic data related to drug absorption, metabolism, and toxicity. In recent years there have been rapid developments in cell-based assays in microfluidic systems, particularly in the area of lab-on-a-chip, enabling the efficient analysis of complex biological phenomenon within microscale systems. 1 Clearly, the generation of quantitatively reliable information onchip, which is a true reflection of the cell's response to a drug or an analyte, remains an important challenge within the biopharmaceutical industry.To date, microfluidic devices have been constructed to study either single-cell or population responses to a variety of factors such as exposure to agonists, electrical stimulation, or variations in the solution composition that may be quiescent or flowing. In general, hydrodynamic forces (or shear stresses) are more significant within microfluidic systems, 2 compared to open quiescent microtiter plates. Several studies have already shown that shear stress can have a range of effects dependent on both the cell type and the local hydrodynamic environment. [3][4][5][6][7] For example, the severity of shear stress can influence the metabolism of hepatocytes 3 and the morphology and metabolism of shearsensitive endothelia cells. 6,7 Outside of the microfluidic environment shear stress has also been found to modulate ion channel (i.e., K + , Ca 2+ ) activation in mechanosensitive cell types, such as endothelial cells 8 and bone cells. 9 Furthermore, it has been observed that Ca 2+ flux is modulated in artificially constructed bilayers by changes in shear stress. 10 This latter observation suggests that similar variations in Ca 2+ flux may exist even for cell types that are generally regarded as non-mechano-sensitive, when they are subjected to the fluid flow regimes found in microfluidic devices where moderate to high shear stresses can readily exist.The importance of having an understanding of the fundamental reasons behind variations in cellular-based Ca 2+ flux can be appreciated since Ca 2+ is generally regarded as a ...
In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.
Recent biochemical and molecular approaches have begun to establish the protein interactions that lead to desmosome assembly. To determine whether these associations occur in native desmosomes we have performed ultrastructural localisation of specific domains of the major desmosomal components and have used the results to construct a molecular map of the desmosomal plaque. Antibodies directed against the amino- and carboxy-terminal domains of desmoplakin, plakoglobin and plakophilin 1, and against the carboxy-terminal domains of desmoglein 3, desmocollin 2a and desmocollin 2b, were used for immunogold labelling of ultrathin cryosections of bovine nasal epidermis. For each antibody, the mean distance of the gold particles, and thus the detected epitope, from the cytoplasmic surface of the plasma membrane was determined quantitatively. Results showed that: (i) plakophilin, although previously shown to bind intermediate filaments in vitro, is localised extremely close to the plasma membrane, rather than in the region where intermediate filaments are seen to insert into the desmosomal plaque; (ii) while the ‘a’ form of desmocollin overlaps with plakoglobin and desmoplakin, the shorter ‘b’ form may be spatially separated from them; (iii) desmoglein 3 extends across the entire outer plaque, beyond both desmocollins; (iv) the amino terminus of desmoplakin lies within the outer dense plaque and the carboxy terminus some 40 nm distant in the zone of intermediate filament attachment. This is consistent with a parallel arrangement of desmoplakin in dimers or higher order aggregates and with the predicted length of desmoplakin II, indicating that desmoplakin I may be folded or coiled. Thus several predictions from previous work were borne out by this study, but in other cases our observations yielded unexpected results. These results have significant implications relating to molecular interactions in desmosomes and emphasise the importance of applying multiple and complementary approaches to biological investigations.
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