Bio-Rad Bio-Plex Pro™ Rat Cytokine, Chemokine, and Growth Factor Assays - Manual (Page 25)

23
Add Coupled Beads, Standards, and Samples
1. Cover unused wells with sealing tape.
2. Prewet the filter plate. Skip this step if using a flat-bottom plate.
Prewet the wells using 100 µl assay buffer and remove the liquid
by vacuum filtration. Dry the bottom of the filter plate thoroughly by
blotting on a clean paper towel.
3. Vortex the diluted (1x) coupled beads for 30 sec at medium speed.
Pour the diluted coupled beads into a reagent reservoir and transfer
50 µl to each well of the assay plate.
Tip: A multichannel pipet is highly recommended for ease of use
and efficiency.
4. Wash the plate two times with 100 µl Bio-Plex ® wash buffer using
the wash method of choice.
5. Gently vortex the diluted standards, blanks, samples, and controls
(if applicable) for 5 sec. Transfer 50 µl to each well of the assay plate,
changing the pipet tip after every volume transfer.
6. Cover plate with a new sheet of sealing tape and protect from light
with aluminum foil. Incubate on shaker at 850 ± 50 rpm at room
temperature (RT). See Table 16 for incubation time.
Note: 850 rpm provides equivalent performance to shaker settings
recommended in previous manuals (1,100 rpm for 30 sec, 300 rpm
for incubation).
Table 16. Sample incubation times.
Assay
Incubation Time
Bio-Plex Pro human cytokine (group I and II) 30 min
Bio-Plex Pro mouse cytokine (group I and II) 30 min
Bio-Plex Pro mouse cytokine (group III) 1 hr
Bio-Plex Pro rat cytokine (group I) 1 hr
Note: Be consistent with this incubation time for optimal assay performance and reproducibility.