Schematic illustration of the fabrication of the microfluidic device.
Operation of the microchip to generate stepwise gradient. (a) Schematic illustration of the operation. Valve channels (groups 1–3) are shown in red (valve is closed) and pink (valve is open). The fluidic channels are in the shape of a ladder (blue, gray, and black colors stand for different solutions). [(b)–(d)] Generation of stepwise gradient of pH in the microchambers. Crystal violet (a pH indicator) is added into the solution to visualize the formed pH values. The lower three figures in (a) correspond to (b), (c), and left half of (d), respectively.
Quantification of fluorescein concentrations in the microfluidic chambers. (a) Fluorescent intensity of fluorescein solution in the microchamber changes linearly with its concentration. (b) Comparison of measured fluorescein concentrations in the microchambers and calculated values of concentrations. The calculated values are obtained by multiplying the original fluorescein solution concentration with dilute factors, which are determined by the chip design.
Representative phase contrast and fluorescent images of HeLa-C3 cells in a typical microchamber treated with etoposide at different time points. [(A)–(E)], [(a)–(e)], and show the phase contrast, YFP fluorescent, and CFP fluorescent images, respectively. The line-shaped shadow in the phase contrast images is from the diffraction of light at two sides of the microchamber, which are curved under the influence of the valve control channels.
Single-cell apoptosis analysis. [(a)–(c)] show the monitoring of apoptotic process of five individual cells in three (low, medium, and high) etoposide concentrations at 9, 55, and , respectively.
(a) Fluorescent image of cell nuclear DNA and (b) phase contrast image of morphology of HeLa-C3 cells. The line-shaped shadow in the phase contrast images is from the diffraction of light at two sides of the microchamber, which are curved under the influence of the valve control channels.
Averaged results of on-chip apoptosis assay. The relative Y/C ratio from YFP and CFP fluorescence images of each cell in all microchambers was recorded every 12 h for 2 days and averaged to give the relative Y/C ratio for each concentration at each time point. The number of individual cells recorded at each concentration is .
Other assay methods of detection of apoptosis. (a) Caspase-3 activity assay with a 96-well plate. (b) MTT assay (the viability is relative to the control). All results are obtained from plate readers.
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