Multiplexing

mirrorball^® is the first system in its class to offer simultaneous scanning with multiple lasers.  When combined with multi-colour fluorescence detection, mirrorball can achieve high levels of multiplexing in microplate bead- and cell-based applications.

Multiplexing options:

• Detection fluorophores – select from 405 nm, 488 nm and 640 nm excitable reagents
• Beads – encode with different fluorophores or level of fluorophore content
• Cells – apply stains to distinguish cell types or report biological activity

A common approach for antibody discovery is the screening of libraries against parental and transfected cell lines to identify antigen-specific binding.  Normally, this requires the running of separate assays for each cell line, however, mirrorball's multiplexing capabilities allows tests to be performed in a single well. This has several advantages including elimination of inter-plate variability, increased throughput and reduced reagent usage.

To illustrate the approach, A549 (EGFR+) and Jurkat (EGFR-) cells were screened for anti-EGFR antibody binding. Jurkat cells were stained with carboxyfluorescein (CFSE) prior to assay. Mirrorball’s unique ability to scan concurrently with multiple lasers allows differentiation of A549 and Jurkat cells (CFSE; 488 nm) based on CFSE staining and simultaneous quantitation of anti-EGFR binding (Alexa 647; 640 nm).

Chart shows a comparison of single and multiplexed assays.

Figure shows 3D fluorescence intensity profiles of A549 and Jurkat cells for 100 ng/mL anti-EGFR antibody.  Note CFSE staining of Jurkat and positive anti-EGFR binding to A549.