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Smooth muscle cell (SMC) heterogeneity plays an important role in vascular remodeling, a life-threatening hallmark of many vascular diseases. However, the characterization of SMCs at the single-cell level is stymied by drawbacks of contemporary single-cell protein measurements, including antibody probe cross-reactivity, chemical fixation artifacts, limited isoform-specific probes, low multiplexing and difficulty sampling cells with irregular morphologies. To scrutinize healthy vessels for subpopulations of SMCs with proliferative-like phenotypes, we developed a high-specificity, multiplexed single-cell immunoblotting cytometry tool for unfixed, uncultured primary cells. We applied our assay to demonstrate maturation stage profiling of aortic SMCs freshly isolated from individual mice. After ensuring unbiased sampling of SMCs (80-120 μm in length), we performed single-SMC electrophoretic protein separations, which resolve protein signal from off-target antibody binding, and immunoblotted for differentiation markers α-SMA, CNN-1 and SMMHC (targets ranging from 34 kDa to 227 kDa). We identified a subpopulation of immature-like SMCs, supporting the recently-established mechanism that only a subset of SMCs is responsible for vascular remodeling. Furthermore, the low sample requirements of our assay enable single-mouse resolution studies, which minimizes animal sacrifice and experimental costs while reporting animal-to-animal phenotypic variation, essential for achieving reproducibility and surmounting the drawbacks of pooling primary cells from different animals.

Mouse-to-mouse variation in maturation heterogeneity of smooth muscle cells