Luminex 100 IS Version 2.2 - Manual (Page 83)

PN 89-00002-00-032 Rev. A 77
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 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.
Microsphere separation
methods
Luminex assay development often requires separation of
microspheres from an aqueous matrix.
Centrifugation. The 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 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.
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.
Microsphere stability and
storage
Luminex 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.