Bio-Rad Rat Diabetes Assays - Manual (Page 22)

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6. Prepare Coupled Beads
Instructions are provided for diluting the coupled beads to a 1x
concentration. When mixing diabetes and cytokine assays, keep in mind
the stock concentrations of coupled beads as listed in Table 9.
Note: When using 10-pack reagents, ensure that only the required
volumes of coupled beads, detection antibodies, streptavidin-PE, and
buffers have been removed from the tubes or bottles. For example, transfer
a one-time volume of assay buffer sufficient to perform all steps of the
assay procedure (that is, prewetting the filter plate, diluting coupled beads,
diluting streptavidin-PE, and resuspending the beads) into a 50 ml reservoir.
1. Use Tables 10–12 to calculate the volume of coupled beads and
assay buffer needed.
2. Add the required volume of Bio-Plex assay buffer to a 15 ml
polypropylene tube.
3. Vortex the stock coupled beads at medium speed for 30 sec.
Carefully open the cap and pipet any liquid trapped in the cap back
into the tube. This is important to ensure maximum bead recovery.
Do not centrifuge the vial; doing so will cause the beads to pellet.
4. Dilute coupled beads to 1x by pipetting the required volume into the
15 ml tube. Vortex.
Each well of the assay plate requires either 2.5 µl (20x stock) or 5.0 µl
(10x stock) adjusted to a final volume of 50 µl in assay buffer.
Assay Stock Concentration of Coupled Beads
Human/NHP, mouse, and rat diabetes 20x
Human and mouse cytokines (groups I, II) 10x
Rat cytokines (group I) 20x
Mouse cytokines (group III) 20X
Table 9. Stock concentration of coupled beads.