Bio-Rad Bio-Plex Pro™ Human Th17 Cytokine Assays - Manual (Page 20)

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Table 8. Detailed wash and incubation instructions.
Add Coupled Beads, Samples, Standards, Blank,
and Controls
1. Cover unused wells of the assay plate with sealing tape.
2. Prewet the filter plate. Skip this step if using a flat-bottom plate.
a) Vacuum manifold: Prewet the wells with 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.
WASH
After each assay step, select the appropriate Bio-Plex Pro ™ wash station program, or perform the
appropriate manual wash step as summarized below.
Using the Bio-Plex Pro Series Wash Stations
For detailed instructions, please refer to the quick guide (bulletin 5826) or user manual (item
#10013125). Both documents can be found at www.bio-rad.com/bio-plex.
Using the Bio-Plex
® Handheld Magnetic Washer
For detailed instructions, please refer to the user guide (item #10023087), which can be
found at www.bio-rad.com/bio-plex.
Using a Vacuum Manifold
Place the filter assay plate on a calibrated vacuum apparatus and remove the buffer by
vacuum filtration. Add 100 μl wash buffer to each well and remove the liquid as before.
Ensure that all wells are exposed to the vacuum. As a general guideline, 100 μl should take
approximately 3–4 sec to completely clear the well. Repeat as specified. Thoroughly blot
the bottom of the filter plate with a clean paper towel between each vacuum step to prevent
cross contamination and plate leakage. Place the assay plate on the plastic plate holder.
COVER AND INCUBATE
Gently cover the plate with a new sheet of sealing tape. To prevent cross-well contamination
or leaking from the bottom of filter plates, do not press down on the tape. Place the plate on a
microplate shaker and cover with aluminum foil to prevent photobleaching. Vigorous shaking
is important. Shake at room temperature at 850 ±50 rpm for the specified incubation time.
Slowly ramp up to the maximum speed to avoid splashing of samples.
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