Bio-Rad Apoptosis Assays - Manual (Page 4)

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Principle
Technology
The Bio-Plex ® multiplex system is built upon the three core elements of
xMAP technology:
n
Fluorescently dyed magnetic microspheres (also called beads), each
with a distinct color code or spectral address to permit discrimination of
individual tests within a multiplex suspension. This allows simultaneous
detection of up to 500 different molecules in a single well of a 96-well
microplate on the Bio-Plex
® 3D system, up to 100 different molecules
on the Bio-Plex ® 200 system, and up to 50 different molecules on the
Bio-Plex ® MAGPIX ™ system
n
A dedicated plate reader. The Bio-Plex 200 and Bio-Plex 3D systems
are flow cytometry–based instruments with two lasers and associated
optics to measure the different molecules bound to the surface of the
beads. In the Bio-Plex MAGPIX system, the entire sample load volume
is injected into a chamber where the beads are imaged using LED and
CCD technology
n
A high-speed digital signal processor that efficiently manages the
fluorescence data
Assay Format
The Bio-Plex Pro ™ RBM apoptosis assays are essentially immunoassays
formatted on magnetic beads. The assay principle is similar to that of a
sandwich ELISA (Figure 1). Capture antibodies directed against the desired
biomarker are covalently coupled to the beads. Coupled beads react with
the sample containing the biomarker of interest. After a series of washes
to remove unbound protein, a biotinylated detection antibody is added to
create a sandwich complex. The final detection complex is formed with
the addition of streptavidin-phycoerythrin (SA-PE) conjugate. Phycoerythrin
serves as a fluorescent indicator, or reporter.