Bio-Rad Apoptosis Assays - Manual (Page 21)

Table 7. Summary of wash options and protocols. 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
n
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 plate seal.
Avoid pressing down on the wells to prevent leaking from the bottom
Assay Protocol: Dispensing of Reagents
1. Add 10 µl of blocker to all wells of the plate.
2. Add 30 µl of the standard, control, or sample to the appropriate well of
the plate.
3. Vortex the capture beads at medium speed for 10–20 sec. Add 10 µl of
the beads to all wells of the plate.
4. Cover plate with plate seal and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 1 hr at RT.
5. Wash the plate three times with 100 µl 1x assay buffer.
6. Vortex the reconstituted detection antibodies at medium speed for
10–20 sec. Add 40 µl to each well.
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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
Sample incubation
MAG x3 VAC x3 3 x 100 μl
SA-PE incubation