Bio-Rad Bio-Plex Pro™ Human Chemokine Assays - Manual (Page 21)

19
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.
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.
3. Vortex the diluted (1x) beads for 30 sec at medium speed. Pour 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 per
well, using the wash method of choice.
5. Vortex the diluted samples, standards, blank, and controls at medium
speed for 5 sec. Transfer 50 µl of each to the appropriate 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 for 1 hr at RT.
Note: Be consistent with this incubation time and shaker setting for
optimal assay performance and reproducibility.
Prepare and Add Detection Antibodies
1. While the samples are incubating use Tables 9 and 10 or the
Calculation Worksheet on page 36 to calculate the volume of
detection antibodies and Bio-Plex detection antibody diluent HB
needed to prepare a 1x stock. Detection antibodies should be
prepared 10 min before use.
2. Add the required volume of Bio-Plex detection antibody diluent HB to
a 15 ml polypropylene tube.
3. Vortex the 20x stock of detection antibodies for 15–20 sec at
medium speed, then perform a 30 sec spin to collect the entire
volume at the bottom of the tube.