Programmed Cell-Death Mechanism Analysis Using Same-Cell, Multimode DNA and Proteoform Electrophoresis.

Gaining insight into the timing of cell apoptosis events requires single-cell-resolution measurements of cell viability. We explore the supposition that mechanism-based scrutiny of programmed cell death would benefit from same-cell analysis of both the DNA state (intact vs fragmented) and the protein states, specifically the full-length vs cleaved state of the DNA-repair protein PARP1, which is cleaved by caspase-3 during caspase-dependent apoptosis. To make this same-cell, multimode measurement, we introduce the single-cell electrophoresis-based viability and protein (SEVAP) assay. Using SEVAP, we (1) isolate human breast cancer SKBR3 cells in microwells molded in thin polyacrylamide gels, (2) electrophoretically separate protein molecular states and DNA molecular states-using differences in electrophoretic mobility-from each single-cell lysate, and (3) perform in-gel DNA staining and PARP1 immunoprobing. Performed in an open microfluidic device, SEVAP scrutinized hundreds to thousands of individual SKBR3 cells. In each single-cell lysate separation, SEVAP baseline-resolved fragmented DNA from intact DNA (