Bio-Rad Bio-Plex Software® Upgrades and Conversions - Manual (Page 242)

Bio-Plex Manager Software 6.1 User Guide | Appendix
230
Enumeration of Microsphere Suspensions
xMAP microspheres are provided at standard concentrations. The microsphere
yield after a coupling process may be less than the starting concentration due to
loss during wash steps. The 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 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 hemocytometer.
Please follow counting and calculation methods outlined in the instructions for
your hemocytometer.
Microsphere Separation Methods
Bio-Plex assay development often requires separation of microspheres from
an aqueous matrix. This can be done using either centrifugation or vacuum
methods.
C ENTRIFUGATION
Microsphere coupling protocols require separation of microspheres from
reaction mixtures or wash buffers by means of centrifugation. Generally, a
tabletop microcentrifuge that can spin 1.5 ml microcentrifuge tubes can pellet
microspheres in 1-2 minutes. The g-forces vary in these instruments from
approximately 6,000 to 13,000 X g.
V ACUUM
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.
BioPlex_6.book Page 230 Friday, September 23, 2011 11:55 AM