Luminex xPONENT 3.1 - Manual (Page 16)

Luminex xPONENT 3.1 Software Manual
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each set of xMAP beads used for a particular assay will show a statistically even
distribution of reporter molecules bound to the surface of each bead. During data
acquisition, numerous beads of each set are analyzed and the median statistic is
computed for that set by the software. The more beads of a set measured, the more
confidence that can be given for that particular measurement. If running an xMAP-based
kit, follow the kit’s product insert or use the provided software protocol.
Classification and Reporter Fluorochromes
Each xMAP bead set is internally dyed with two classification dyes. The fluorescence
signal of these dyes allows for classification of each bead set. Since each bead is
analyzed individually, even when the sets are mixed in a multiplex assay they can still be
distinguished by their emission signals. The fluorescence signal of reporter molecules
bound to the surface of each bead set is measured and used to determine the result of
each assay in a multiplex. Again, since each bead is analyzed individually, reporter signals
for each bead set can be accurately quantified.
The following table displays acceptable reporter fluorochromes and their excitation and
emission wavelengths.
Sample Volume
Sample volumes or sample sizes range from 10 to 200 µL. Ensure that some sample
remains in the well after aspiration; about 25 µL greater than the sample volume. This
amount may vary depending on the type of plate used. After acquisition, the Luminex
analyzer washes the sample lines resulting in ejection of approximately 165 µL of sheath
fluid back into the well for a 96-well plate. Ensure that there is room to add this amount to
the well without overflowing and contaminating other wells.
The volume restrictions on the assay design can be expounded by the following formula:
Total well volume (µL) – Sample uptake volume (µL) + 165 (µL) <Maximum Well Volume
(µL)
• Total well volume = Starting sample volume of a well before the unit samples for
acquisition. Well volume is determined by the consistency of the bead set.
• Sample uptake volume = Uptake volume for acquisition (program this in the protocol as
sample volume).
• 165 (µL) = Volume expelled back as stated in the above paragraph.
• Maximum well volume plate = The maximum volume capacity of the wells in a selected
96-well microtiter plate.
TABLE 1. Reporter Fluorochromes Wavelengths
R-Phycoerythrin Alexa 532
Formula weight (Daltons) 240,000 470
Absorbance max (nm) 480, 546, 565 531
Extinction max (M-1cm-1) 1,960,000 83,800
Emission max (nm) 578 554
Quantum yield 0.82 0.8