Bio-Rad Bio-Plex Pro™ Human Isotyping Assays - Manual (Page 19)

8. Run Assay
Considerations
n
Bring all buffers, diluents, diluted standards, diluted coupled beads, and
samples to room temperature before use
n
Use calibrated pipets and pipet carefully, avoiding bubbles. Use new
pipet tips for every volume transfer
n
Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from the protocol may result in low assay signal and
assay variability
n
Assay incubations are carried out in the dark at 850 ± 50 rpm. Cover
the plate with sealing tape and protect from light with aluminum foil
Table 7. Summary of wash steps and incubations. After each assay step, select the
appropriate Bio-Plex Pro ™ wash station program or perform the appropriate manual wash step
as summarized below.
Considerations When Using a Vacuum Manifold
n
After each incubation, place the filter plate on a calibrated vacuum
apparatus and remove the liquid by vacuum filtration
n
To wash, add 100 μl wash buffer to each well and remove the liquid as
before. Ensure that all wells are exposed to the vacuum
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Thoroughly blot the bottom of the filter plate with a clean paper towel
between each vacuum step to prevent cross contamination
n
Place the assay plate on the plastic plate holder/tray as needed
n
Before each incubation, gently cover the plate with a new sheet of sealing
tape. Avoid pressing down over the wells to prevent leaking from the bottom
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Bio-Plex Pro or Bio-Plex Pro II Handheld Magnet or
Pro II Wash Station Wash Station Vacuum Manifold
Assay Step Magnetic Program Vacuum Program Manual Wash Steps
Add beads to plate MAG x2 VAC x2 2 x 100 μl
Sample incubation
Detection Ab incubation
MAG x3 VAC x3 3 x 100 μl
SA-PE incubation