Fig 1. Hoechst Staining Overview and SP Assay Conceptual Model.

(A) Experimental workflow of sample preparation and processing in a SP assay. Each sample (1) is split into two conditions, with (+inhibitor) and without (-inhibitor) the ABC transporter inhibitor (2). Both conditions are then stained with Hoechst 33342 (3) and the resultant fluorescence measured via flow cytometry (4). The +inhibitor condition is used to define a gate for the non-side population (NSP) region (5), which is then applied to the -inhibitor condition (6) to identify the SP region (green box), which is measured as percent of the parent population (7). (B) Schematic of the Hoechst transport processes presumed to underlie the SP assay. Hoechst 33342 passively diffuses into the cell, where it is transported out of the cell via transporter (-inhibitor condition) or binds to DNA. For the +inhibitor condition, a small molecule compound inhibits the transporter, preventing transporter-mediated efflux. (C) Hoechst staining dynamics were simulated at the single-cell level with each cell represented by a set of ODEs governed by mass-action kinetics in a well-mixed three-compartment system. The species, compartments, and reactions are depicted. Each cell differs from the rest of the population in terms of volumes, surface areas, transporter properties, and DNA content. Within a given population, all the cells share a common set of kinetic parameters (k).