BIO RAD Bio-Plex Quick Manual
Bio-Plex Pro Human Immunotherapy Panel
Quick Guide
For Use wi th Inst ruction Manu al #
Bio-Plex Pro Human Immunotherapy Assays 10000112563
This guide can be used to prepare and run a full 1 x 96-well assay plate. New
users can download the complete manual, which includes detailed instructions
and a list of kit components, at bio-rad.com/bio-plex.
Initial Preparation
1. Plan the plate layout.
2. Start up/warm up the Bio-Plex Multiplex Immunoassay System (30 min).
■
Bring diluents, including wash buffer, assay buffer, standard
diluent HB, detection antibody diluent HB, and sample diluent HB,
to room temperature (RT). Keep the other items on ice until needed
– Mix by inversion to ensure all salts are in solution
– Prepare 1x wash buffer: dilute 1 part 10x wash buffer (60 ml) with
9 parts distilled water (540 ml)
■
Begin to thaw the frozen samples
3. Prepare the sample dilution according to the guidelines provided in the
following table. It is important to centrifuge serum or plasma samples at
1,000 x g for 15 m in at 4°C to remove particulates from all samples prior to use.
Sample Type Recommended Sample Dilution Diluent
Serum and plasma 1:4 Sample diluent
Culture media and uids User dened Diluent + 0.5% bovine
Note: ICAM-1 and VCAM -1 require higher di lution for ser um and pl asma (re comm ended 100-fold ). Refer
to the Bio-Plex Pro Human Immunotherapy Assays Instruction Manual (#10000112563) for detailed sample
preparation recommendations.
serum albumin (BSA) (w/v)
4. Calibrate the Bio-Plex System within Bio-Plex Manager Software.
5. Reconstitute the standards and control by adding 250 µl of standard
diluent HB to each. Vortex at medium speed for 5 sec and incubate all
vials on ice for precisely 30 min.
Bio-Plex Pro Human Immunotherapy Panel Quick Guide
Standard Serial Dilution
6. Prepare a fourfold standard dilution series and blank as shown.
Vortex at medium speed for 5 sec between liquid transfers.
Note: Standards are at S1 concentration after reconsititution and the
controls are ready to use after reconstitution. Controls are included with
the xed panel only.
Transfer Volume, µl
Reconstituted
Standard
Standard Diluent, µl
50250 50 50 50 50 50 50
S1
S2 S3 S4 S5 S6 S7 S8 Blank
1500 150 150 150 150 150 150 150
7. Vortex the coupled beads at medium speed for 30 sec and dilute to 1x
in Bio-Plex Assay Buffer as shown. Protect from light.
Premixed Panels
Number of Wells 10x Beads, µl Assay Buffer, µl Total Volume, µl
96 570 5,1 3 0 5,700
Singleplex Assays
Singleplex #1
Number of Wells
96 285 285 5,13 0 5,700
Note: 20x sing lepl ex beads allow mu ltipl exing up to 20 analy tes.
20x Beads, µl
Singleplex #2
20x Beads, µl Assay Buffer, µl Total Volume, µl
Running the Assay
1. Vortex the diluted (1x) beads. Add 50 µl to each well of the assay plate.
2. Wash the plate two times with 100 µl Bio-Plex Wash Buffer.
3. Vortex the samples, standards, blank, and control. Add 50 µl to each well.
4. Cover the plate with sealing tape. Incubate on shaker at 850 ± 50 rpm at
RT for 30 min.
5. With 10 min left in the incubation, vortex the detection antibodies for 5 sec
and quick-spin to collect liquid. Dilute to 1x as shown.
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