Bio-Rad Bio-Plex Pro™ Human Th17 Cytokine Assays - Manual (Page 21)

19
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 wells twice with 100 µl Bio-Plex wash buffer according to
your wash method of choice.
5. Vortex the diluted samples, standards, blank, and controls 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 and incubate in the dark for 1 hour at room temperature (RT)
with shaking at 850 ±50 rpm.
Prepare and Add Detection Antibodies
1. While the samples are incubating use Tables 9–10 or the Calculation
Worksheet on page 35 to calculate the volume of detection
antibodies and Bio-Plex detection antibody diluent needed. These
calculations include ~20% excess to compensate for transfer loss.
Detection antibodies should be prepared 10 min before use.
2. Add the required volume of Bio-Plex detection antibody diluent 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.
4. Dilute detection antibodies to 1x by pipetting the required volume into
the 15 ml tube. Vortex.
Each well of the assay requires 1.25 μl of the 20x stock adjusted to a
final volume of 25 μl in detection antibody diluent.