Luminex 100 IS Version 2.1 - Manual (Page 73)

PN 89-00002-00-062 Rev. A 67
x MAP Technology
It is not advisable to construct an assay without defining the
concentration of the subject-coupled microsphere preparation. The
total surface area (total number of xMAP microspheres) represented
in an assay is a critical variable, so the optimization and control of
this variable begins with manipulation of known microsphere
numbers. xMAP microspheres can be counted with a hemacytometer.
Please follow counting and calculation methods outlined in the
instructions for your hemacytometer.
xMAP Microsphere
Separation Methods
Luminex assay development often requires separation of
microspheres from an aqueous matrix.
Centrifugation. The xMAP microsphere coupling protocols
provided in this guide require separation of xMAP microspheres
from reaction mixtures or wash buffers by means of centrifugation.
Generally, a table-top microcentrifuge that can spin 1.5 mL
microcentrifuge tubes can pellet Luminex microspheres in 1 to 2
minutes. The g-forces vary in these instruments from approximately
6,000 to 13,000
× g.
Vacuum. A vacuum separation method can be used for routine high
throughput washing required for washed assay procedures. In this
case, the microsphere reactions are performed in microtiter filter-
bottom 96-well plates. A vacuum is applied to the plate allowing the
liquid to filter through while retaining the microspheres on the filter.
Resuspension is accomplished by adding adequate fluid to each well
and by repetitive, vigorous pipetting.
xMAP Microsphere
Agitation During Assay
Agitation of Luminex assays with a plate shaker during incubation
steps prevents microspheres from settling during extended incubation
periods. The benefit of agitation may not be discernible for some
assay conditions and should be evaluated according to the
requirements of a given application.
xMAP Microsphere
Stability and Storage
Luminex xMAP microspheres are light sensitive and should be
protected from light during all stages of storage and usage. In
addition, freezing conditions and organic solvents should be avoided.