Microscopy often reveals the existence of phenotypically distinct cellular subpopulations. However, additional characterization of observed subpopulations can be limited by the number of biomolecular markers that can be simultaneously monitored. Here we present a computational approach for extensibly profiling cellular subpopulations by freeing one or more imaging channels to monitor additional probes. In our approach, we trained classifiers to re-identify subpopulations accurately based on an enhanced collection of phenotypic features extracted from only a subset of the original markers. Then we constructed subpopulation profiles step-wise from replicate experiments, in which cells were labeled with different but overlapping marker sets. We applied our approach to identify molecular differences among subpopulations and to identify functional groupings of markers, in populations of differentiating mouse preadipocytes, polarizing human neutrophil-like cells and dividing human cancer cells.
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We thank all members of the Altschuler and Wu lab at the University of Texas Southwestern Medical Center for critical discussion and for performing manual cell categorization; P.E. Scherer (University of Texas Southwestern Medical Center) and O.D. Weiner (University of California, San Francisco) for the gifts of the adiponectin and Hem1 antibodies, respectively; J. Rhorer at BD Biosciences for the gift of the cell cycle kit; and S.A. Kliewer, D.J. Mangelsdorf, J. Repa P.E. Scherer and H.T. Yu for stimulating conversations. This work was funded by the US National Institutes of Health (R01 GM081549 to L.F.W. and R01 GM085442 to S.J.A.), the Welch Foundation (I-1619 and I-1644 to L.F.W. and S.J.A.), the Rita Allen Foundation (S.J.A.) and the University of Texas Southwestern Endowment for Scholars in Biomedical Research (to L.F.W. and to S.J.A.). S.J.A. is a Rita Allen Scholar.
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Loo, LH., Lin, HJ., Steininger, R. et al. An approach for extensibly profiling the molecular states of cellular subpopulations. Nat Methods 6, 759–765 (2009). https://doi.org/10.1038/nmeth.1375
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