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 (
R
s
= 5.17) as well as cleaved PARP1
from full-length PARP1 (
R
s
= 0.66). Comparing
apoptotic and viable cells showed statistically similar profiles (expression,
mobility, peak width) of housekeeping protein β-tubulin (Mann–Whitney
U test). Clustering and cross-correlation analysis of DNA migration
and PARP1 migration identified nonapoptotic vs apoptotic cells. Clustering
analysis further suggested that cleaved PARP1 is a suitable apoptosis
marker for this system. SEVAP is an efficient, multimode, end-point
assay designed to elucidate cell-to-cell heterogeneity in mechanism-specific
signaling during programmed cell death.
Protein isoforms play a key role in disease progression and arise from mechanisms involving multiple molecular subtypes, including DNA, mRNA and protein. Recently introduced multimodal assays successfully link epigenome, genomes...
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