Luminex 100 User Manual Version 1.7 - Manual (Page 113)

x MAP Technology Protocols
PN 89-00002-00-063 Rev. A 9 - 3
Enumeration of xMAP
Microsphere Suspensions
Luminex xMAP microspheres are provided at standard
concentrations. The xMAP microsphere yield after a coupling
process may be less than the starting concentration due to loss during
wash steps. The xMAP microsphere loss can vary according to
operator technique, coupled reactant properties, and scale of
coupling.
It is not advisable to construct an assay without defining the
concentration of the coupled microsphere suspension. The total
surface area (total number of microspheres) represented in an assay
is a critical variable, so the optimization and control of this variable
begins with manipulation of known microsphere numbers.
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 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 xMAP 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 xMAP 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 xMAP
microspheres on the filter. Resuspension is accomplished by adding
adequate fluid to each well and by repetitive, gentle pipetting up and
down five times. You do not need to vacuum the plate completely
dry.
xMAP Microsphere
Agitation During Assay
Gentle agitation of Luminex assays with a plate shaker during
incubation steps prevents xMAP 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.