Bio-Rad Rat Diabetes Assays - Manual (Page 29)

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5. After the detection antibody incubation, slowly remove and discard
the sealing tape.
6. Wash the plate three times with 100 µl wash buffer.
7. Vortex the diluted (1x) SA-PE at medium speed for 5 sec. Pour into a
reagent reservoir and transfer 50 µl to each well using a multichannel
pipet.
8. Cover plate with sealing tape and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 10 min at room temperature.
9. After the streptavidin-PE incubation step, slowly remove and discard
the sealing tape.
10. Wash the plate three times with 100 µl wash buffer.
11. To resuspend beads for plate reading, add 125 µl of assay buffer to
each well. Cover the plate with a new sheet of sealing tape. Shake
the plate at room temperature at 850 ± 50 rpm for 30 sec, and
slowly remove the sealing tape. Ensure that the plate cover has been
removed before placing the plate on the reader.
12. Remove the sealing tape and read the plate using the settings below.
Note: Reading at alternative PMT settings on the Bio-Plex 100,
200, or Bio-Plex 3D (for example when mixing diabetes assays with
cytokine assays) requires validation by the end user to ensure that
results meet the user’s acceptance criteria.
Instrument RP1 (PMT) DD Gates Bead Events
Bio-Plex 100, 200* High 5,000 (low), 25,000 (high) 50
Bio-Plex 3D* Standard Select MagPlex beads 50
Bio-Plex ® MAGPIX ™ * N/A, use default instrument settings
* Or similar Luminex-based system.
Table 19. Read the plate using the appropriate instrument settings.