Bio-Rad Bio-Plex Pro™ Magnetic Cell Signaling Assays - Manual (Page 21)

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Preparing 1x detection antibodies from 20x stock (includes 25% excess volume):
Table 8. Premixed panel or one singleplex assay.
Table 9. Mixing two singleplex assays.
22. After the overnight incubation, slowly remove and discard the
sealing tape.
23. Wash the plate three times with 200 µl wash buffer according to
your method of choice.
24. Vortex the diluted (1x) detection antibodies gently for 5 sec and
transfer 25 µl to each well of the assay plate.
25. Cover with a new sheet of sealing tape and incubate in the dark for
30 min at room temperature with shaking. Fully resuspend the beads/
detection antibody mixture by shaking at 900–1,100 rpm for 30 sec.
Then turn down to 300–450 rpm for the specified incubation time.
Prepare and Add Streptavidin-PE (SA-PE)
26. While detection antibodies are incubating, use Table 10 or the
Calculation Worksheet on pages 32–33 to calculate the volume of
SA-PE and detection antibody diluent needed. SA-PE should be
prepared 10 min before use.
20x Detection Detection Antibody
# of Wells Antibodies, µl Diluent, µl Total Volume, µl
96 150 2,850 3,000
48 75 1,425 1,500
20x Detection 20x Detection Detection
Antibodies, µl Antibodies, µl Antibody
# of Wells Singleplex #1 Singleplex #2 Diluent, µl Total Volume, µl
96 150 150 2,700 3,000
48 75 75 1,350 1,500