High-throughput single-cell fate potential assay of murine hematopoietic progenitors in vitro

The advent of single-cell transcriptomics has led to the proposal of a number of novel high-resolution models for the hematopoietic system. Testing the predictions generated by such models requires cell fate potential assays of matching, single-cell resolution. Here we detail the development of an in vitro high-throughput single-cell culture assay using flow cytometrically sorted single murine bone marrow progenitors, which measures their differentiation into any of five myeloid lineages. We identify critical parameters for single-cell culture outcome, including the choice of sorter nozzle size and pressure, culture media, and the coating of culture dishes with extracellular matrix proteins. Further, we find that accurate assay readout requires the titration of antibodies specifically for their use under low-cell-number conditions. Our approach may be used as a template for the development of single-cell fate potential assays for a variety of blood cell progenitors.