Multiplexing to the Max

A Guide to Stripping and Reprobing Your Single-Cell Westerns

At the core of cell-to-cell heterogeneity are the wide-ranging patterns of protein expression and activity that are responsible for the diversity in individual cell behavior and response. Understanding this variability is critical to understanding the complexity of human biology and the advancement of biomedical research.

More and more, researchers need information about multiple proteins at once. Cell-signaling pathways involve multiple key proteins at any given moment, and cellular responses to novel compounds often include several proteins. Also, the diagnosis of various diseases depends on the status of a handful of proteins. Other single-cell approaches, like flow cytometry, rely on tagged antibodies for multiplexing and fluorescent antibody spectral-crosstalk can limit the maximum multiplexing level to approximately 12 colors or targets.

Single-Cell Westerns with Milo™ allow you to unlock the single-cell proteome and measure changes in protein expression heterogeneity that aren’t possible with other techniques. This technical note will walk you through multiplexing with Milo and provide you with evidence of and guidance for efficient stripping to achieve multiplexing levels that match or exceed those of the most powerful multiplexed flow cytometers.