Bio-Rad Bio-Plex Pro™ Human Chemokine Assays - Manual (Page 27)

25
b. Enter a dilution factor of 4 and click Calculate. The concentrations
for each standard point will be populated for all analytes in the table.
c. optional: enter the lot number of the vial of standards into the
Standard Lot box and click Save.
5. Click Enter Controls Info.
a. For user-specified controls, select an analyte from the dropdown
menu, then enter a description and concentration. Repeat for
each additional analyte in the assay.
b. For the quality controls supplied with the 40-plex fixed panel
only, format the appropriate wells as controls, enter descriptions,
but leave the concentrations blank. Alternatively, the quality
controls can be formatted as samples with clear descriptions
such as “quality control high” and “quality control low.” In any
case, the expected control ranges provided are not entered
into Bio-Plex Manager software version 6.1 and earlier.
6. Click Enter Sample Info and enter sample information and the
appropriate dilution factor.
7. Click Run Protocol and confirm that the assay settings are correct.
a. Refer to Table 12 for the recommended RP1 (PMT) setting.
Protocols using alternative PMT settings should be validated by
the end user.
b. Confirm that data acquisition is set to 50 beads per region.
In Advanced Settings, confirm that the bead map is set to
100 region, the sample size is set to 50 µl, and the doublet
discriminator (DD) gates are set to 5,000 (Low) and 25,000 (High).
In Bio-Plex Manager software versions 4.0, 4.1, 4.1.1, and 5.0,
check Override Gates and set the DD gate values as indicated.
Select Start, name and save the .rbx file, and begin data
acquisition. The Run Protocol pop-up screen will appear. Click
Eject/Retract to eject the plate carrier.