Bio-Rad Bio-Plex Pro™ Rat Cytokine, Chemokine, and Growth Factor Assays - Manual (Page 3)

Running the Assay
1. Prewet filter plate with 100 µl Bio-Plex assay buffer (skip for flat bottom).
2. Vortex the diluted (1x) beads for 10–20 sec. Add 50 µl to each well of
the assay plate.
3. Wash the plate two times with 100 µl Bio-Plex wash buffer.
4. Vortex samples, standards, blank. Add 50 µl to each well.
5. Cover plate with sealing tape and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm at RT. See table for incubation time.
Note: 850 rpm provides equivalent performance to recommended shaker
settings in previous manuals (1,100 rpm for 30 sec, 300 rpm for incubation).
6. With 10 min left in the incubation, vortex the 10x or 20x detection Abs for
5 sec and quick-spin to collect liquid. Dilute to 1x as shown below.
7. Wash the plate three times with 100 µl wash buffer.
8. Vortex the diluted (1x) detection antibodies. Add 25 µl to each well.
9. Repeat Step 5. See table for incubation time. Meanwhile, prepare Bio-Plex
Manager software protocol; enter standard S1 values from the assay kit.
10. With 10 min left in the incubation, vortex the 100x SA-PE for 5 sec and
quick-spin to collect liquid. Dilute to 1x as shown and protect from light.
# of Wells Detection Ab, µl Detection Ab Diluent, µl Total Volume, µl
10x 20x
96 300 — 2,700 3,000
96 — 150 2,850 3,000
Bio-Plex Pro Assay Quick Guide 4
Assay Sample Detection Ab SA-PE
Bio-Plex Pro human and mouse cytokine (groups I and II) 30 min 30 min 10 min
Bio-Plex Pro mouse cytokine (group III) 1 hr 30 min 10 min
Bio-Plex Pro rat cytokine (group I) 1 hr 30 min 10 min
Incubation times for sample, detection Ab, and SA-PE.
# of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl
96 60 5,940 6,000