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Multiplexing strategy to increase assay throughput.

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posted on 28.10.2014, 18:54 by Stella M. Bernardo, Christopher P. Allen, Anna Waller, Susan M. Young, Tudor Oprea, Larry A. Sklar, Samuel A. Lee

A schematic illustrating the amount of red and/or violet dyes used to differentially stain up to 9 different strains is shown in (A). Each sector of the 3×3 block represents one strain to be stained and details the specific concentration and combination of Alexafluor 633 and Alexafluor 405 dyes to be used for multiplexing. A typical sampling from one well carrying equal volumes of each stained cell is shown in a density plot in (B). Individual strains are visualized in a density plot of the two fluorescent channels (FL8 vs FL6), from which data on GFP levels can be extracted. Daughter cells that have lost some or all of the stain migrate to the lower left corner of the histogram. Gating on individual cell populations in the density plots depicted in (B) demonstrate that each population of cells is specific for one strain, with negligible contamination from migrating cells (C).