Mass‐Added Density Modulation for Sorting Cells Based on Differential Surface Protein Levels

Cell sorting is a powerful tool in basic research and therapeutic enrichment. However, common cell sorting methods, such as fluorescence‐activated cell sorting (FACS) and magnetic‐activated cell sorting (MACS) have significant limitations, such as generally low cell yields or restriction to binary separation, respectively. To address these limitations, we developed a two‐step cell sorting method called mass‐added density centrifugation (MADC) to enable nonbinary separation of large cell numbers based on surface protein levels. In the first MADC step (mass‐adding), antibody‐directed massive microparticles bind target surface proteins to modulate single‐cell density proportionally to target protein level. Second, microparticle‐laden cells are subjected to discontinuous density gradient centrifugation, whereby they separate into discrete density bands which can be isolated for downstream use. MADC will prove especially advantageous for obtaining sufficient cell numbers for protein analyses from large source populations, and it is a fast process that can facilitate live cell enrichment for therapies that require tens of millions of cells. Here, we demonstrate MADC's utility for both live and fixed cell sorts of multiple cell types based on abundance of an example target protein, CD44. CD44 quantity in separated cell groups was assayed with western blots and correlated with modulated cell density. This novel sorting method enables rapid, nonbinary isolation of large quantities of cells based on surface protein levels and should prove useful in both basic science and therapeutic applications. © 2020 International Society for Advancement of Cytometry Mass‐added density centrifugation (MADC) enables rapid, high‐throughput cell sorting based on surface protein abundance. Mass‐adding modifies cell density proportionally to protein levels by tagging a protein of interest with antibody‐directed massive microparticles. Density centrifugation then separates cells on a density gradient to sort cells into groups corresponding to protein quantity.