Luminex 100 IS Developer Workbench Guide Version 2.3 - Manual (Page 57)

PN 89-00002-00-084 Rev. B 51
x
MAP Technology xMAP Microsphere Handling
It is not advisable to construct an assay without defining the
concentration of the subject-coupled xMAP 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 xMAP
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 xMAP
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 xMAP microspheres in 1 to 2
minutes. The g-forces vary in these instruments from approximately
6,000 to 20,000
× g.
Vacuum. Use a vacuum separation method 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 gentle 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, vigorous pipetting.
xMAP Microsphere
Agitation During Assay
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.
xMAP Microsphere
Stability and Storage
xMAP microspheres are light sensitive. Protect them from light
during all stages of storage and usage. In addition, avoid freezing
conditions and organic solvents.