Luminex 200 User Manual with LDS 1.7 Software - Manual (Page 15)

x
MAP Technology Introduction
PN 89-00002-00-150 Rev. A 1 - 3
container and effectively removes residual sample within the tubing,
valves, and probe. The second fluidics path is driven under positive
air pressure and supplies sheath fluid to the cuvette.
Excitation
The excitation system in the Luminex analyzer uses two solid-state
lasers. A reporter laser excites fluorescent molecules bound to
biological reactants at the microsphere surface, and a classification
laser excites fluorochromes embedded in the microsphere. The lasers
illuminate the xMAP microspheres as they flow single-file through
the cuvette. These fluorescent signals are discriminated with
selective emission filters and are converted into intensity units by a
digital signal processor.
xMAP Microspheres
The xMAP microspheres are highly uniform, polystyrene particles
that have been crosslinked during polymerization for great physical
and thermal stability. Varying amounts of fluorochromes embedded
within each microsphere give each microsphere set an unique
fluorescent signal. To ensure the stability of this address, it is
essential to protect the xMAP microspheres from light. Do not
subject xMAP microspheres to prolonged high temperatures, and
protect them from freeze/thaw manipulations.
Reporter Fluorochromes
Each xMAP microsphere is dyed to emit light in a certain
classification channel. All xMAP microspheres of a given emission
represent a distinct assay within a multiplex of assays. A reporter
channel is used to detect fluorescence bound to the surface of each
microsphere, and each reporter emission quantitates each of the
distinct assays. Only one reporter emission is used for multi-analyte
assays in the Luminex analyzer. The Luminex analyzer uses an
excitation wavelength of 532 nm, and has an emission wavelength of
575 nm
± 12 nm.
Fluorochromes R-Phycoerythrin and Alexa
®
532 work well with the
Luminex analyzer, but you are not limited to these two. You may
have success using other fluorochromes such as Cyanine 3.
R-
Phycoerythrin
Alexa
532
Formula weight (Daltons) 240,000 470
Absorbance max (nm) 480, 546, 565 531
Extinction max (M
-1
cm
-1
)
1,960,000 83,800
Emission max (nm) 578 554