Browsing by Author "Mao, Chen"

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    Characterization of 3-D collagen hydrogels for functional cell-based biosensing
    (Biosensors and Bioelectronics, 2004) Mao, Chen; Kisaalita, William S.
    To address the growing demand for functional cell-based assay technologies with accelerated drug discovery applications, we have proposed the use of human neuroblastoma cells (IMR-32) immobilized in three-dimensional (3-D) collagen hydrogel matrices. The gel protects weakly adherent cells from fluid mechanical forces while providing a more physiologically relevant 3-D environment. Hydrogels made up of collagen, between 0.5 and 1.0mg/ml, exhibited mechanical stability adequate to withstand fluid mechanical forces (<0.11mN) typical of automated commercial fluid transfer equipment. Collagen-entrapped cells visualized with the aid of confocal microscopy and a potentiometric-sensitive dye, TMRM, exhibited round morphology in comparison to flat morphology typical of cells in two-dimensional (2-D) monolayer cultures. Morphological differentiation characterized by neurite extension and cell aggregation was observed for both 2-D and 3-D cultures. Differentiated IMR-32 cells failed to develop a resting membrane potential typical of excitable cells. Free intracellular calcium was monitored with Calcium Green-1. Depolarization-induced Ca2+ influx was only observed with differentiated 3-D cells unlike 2-D cells, where calcium flux was observed in both differentiated and undifferentiated cells. Taken together, the results revealed that collagen hydrogels (0.5mg/ml collagen) were suitable structural supports for weakly adherent cells. However, for voltage-dependent calcium channel function applications, further investigations are needed to explain the difference between 2-D monolayer and 3-D collagen-entrapped cells
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    Determination of Resting Membrane Potential of Individual Neuroblastoma Cells (IMR-32) Using a Potentiometric Dye (TMRM) and Confocal Microscopy
    (Journal of fluorescence, 2004) Mao, Chen; Kisaalita, William S.
    The potentiometric dye, Tetramethylrhodamine methyl ester (TMRM) has been extensively used with fluorometry or optical microscopy to evaluate the electric potential across plasma or mitochondrial membranes. We present here a TMRM confocal microscopy-based potential measurement technique. Corrections are introduced to minimize nonspecific dye binding and insensitivity to low background levels. We have used this technique to compare the resting membrane potential of proliferating and differentiated human neuroblastoma cells (IMR-32)