Detection of DNA fragmentation in a single apoptotic cardiomyocyte by electrophoresis on a microfluidic device

Abstract

The detection of doxorubicin-induced apoptosis in individual cardiomyocytes was performed on a microfluidic device. Microstructures integrated on a CD-like plastic disk were adapted for the selection of individual cells their lysis in an alkaline environment and the separation of released apoptotic DNA fragments. The fragments with typical 180 base pairs ladder pattern were electrophoretically resolved in a 2% solution of linear polyacrylamide with 0.1 M NaOH on a migration distance of 6 mm. The laser-induced fluorescence of fragments labeled by ethidium bromide was monitored by a photomultiplier tube mounted on a confocal microscope. The causal relation between the enhanced doxorubicin concentration and the extent of DNA fragmentation in a single cell was confirmed. The results show that the extent of DNA fragmentation is proportional to the time of a cell treatment. Onset of necrosis was evident in cardiomyocytes treated by doxorubicin for more than 24 h. The adverse effect of doxorubicin, an important cytostatics used for the treatment of many solid tumors, leads to the destruction of cardiomyocytes and, consequently, may result in the heart failure of treated individuals. Therefore, the monitoring of the extent of apoptotic DNA damage of cardiac myocytes represents critical step toward understanding of the development of chronic doxorubicin-induced cardiomyopathy.

Chemical cytometry on a picoliter-scale integrated microfluidic chip

“…This volume compares favorably with the nanoliter volumes reported for derivatization reactions in capillary-based chemical cytometry methods (1)(2)(3)(4)(5), although smaller volumes have been reported (26). The capacity to confine a lysing, reacting plug of cell and reagents is an advantage that motivated prior work by us (8) and others (18) to use valves formed by multilayer soft lithography to confine approximately nanoliter volumes.…”

Microfluidic single-cell analysis of intracellular compounds

“…As the genotype within pure cell lines is usually identical, it can be argued that single-cell DNA extractions do not provide more information than regular bulk extractions. The work from Kleparnik, however, demonstrated that meaningful information can be gained from such microfluidic devices (Kleparnik & Horky 2003). They used a microfluidic device based on a commercial polycarbonate platform to semi-quantitatively measure DNA fragmentation in mouse cardiomyocytes upon doxorubicin treatment.…”

Microfluidic devices for the analysis of apoptosis

“…Traditional gel electrophoresis method is labor-intensive, requiring an additional time for cell lysing, and extraction of genomic DNA. Kleparnik and Horky [32] performed the detection of doxorubicin-induced apoptosis in individual cardiomyocytes on a microfluidic device. Microstructures integrated on a CD-like plastic disk were adapted for the selection of individual cells, the lysis of cells in an alkaline environment, and the separation of released apoptotic DNA fragments.…”