Bio-Rad Bio-Plex Pro™ Rat Cytokine, Chemokine, and Growth Factor Assays - Manual (Page 15)

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2. Add 500 μl of the appropriate diluent (see Table 5). Do not use assay
buffer to reconstitute the standards.
3. Gently vortex the reconstituted standard for 5 sec then incubate on
ice for 30 min. Be consistent with the incubation time in every
assay to ensure best results.
4. During the incubation period, prepare the samples as instructed in the
Prepare Samples section.
Prepare Standard Dilution Series From a Single
Antigen Vial
The following procedure produces an eight-point standard curve with a
fourfold dilution between each point. Pipet carefully using calibrated pipets
and use new pipet tips for every volume transfer.
1. Label nine 1.5 ml polypropylene tubes S1 through S8 and Blank.
2. Add the specified volume of standard diluent to each tube
(Figures 3 and 4).
3. Vortex the reconstituted standards gently for 5 sec before removing
any volume. Add 128 µl into the S1 tube containing 72 µl of standard
diluent. Vortex at medium speed for 5 sec, then use a new pipet tip
to transfer 50 µl from S1 tube to S2 tube. Vortex.
4. Continue with 1:4 (fourfold) serial dilutions from tube S2 to S8 as
shown in Figure 3. Use reconstituted and diluted standards
immediately. Do not freeze for future use.
Reconstituted
Standard
72 150 150 150 150 150 150 150 150
S1 S2 S3 S4 S5 S6 S7 S8 Blank
Diluent (µl)
128 50 50 50 50 50 50 50 Transfer Volume (µl)
Fig. 3. Preparing a fourfold dilution series of cytokine standards.