B2M

IL-18

TFF3

TNF-a

Osteopontin

KIM-1

VEGF-C
PAI-1

B2M

Clusterin

IL-8

IL-25

IL-17A/F

G-CSF

IFN-g
MCP-1

IL-18
TFF3

KIM-1

The Bio-Plex® Success Guidefor Multiplex Immunoassays
Using Luminex xMAP TechnologyAssay/Sample Prep

May the Plex
Be with You
Episode I: I sense a disturbancewith your assay/sample prep
Not too long ago, in a laboratory not too
far away…
Luminex’s xMAP technology was introduced
to make your experiments faster and more
efficient.
This is a success guide that includes a varietyof tips and tricks for obtaining optimal resultsfrom your Luminex multiplex immunoassays.
(Always follow the product instruction manual
when running an assay.)Serum or Plasma:
What Sample
Do I Choose?

Sample measurements can differ for serum andplasma. Why?
• Serum is qualitatively different from plasma becauseof how the removal of fibrinogen and other proteinsthat form fibrin clots affects coagulation• Protein concentration of serum is ~3–4% less thanthat of plasma
Choose sample matrix measurements that fall within
the linear portion of the standard curve. This willallow for better precision and accuracy.

Avoid serum with gross lipemia or hemolysis.

Steps to a Successful Serum Prep:
1. Allow blood to clot at room temperature for
30–45 min before proceeding to centrifugation
steps.
2. Centrifuge at 1,000 x g for 15 min at 4°C and transfer
into a clean tube.
3. R epeat centrifugation at 10,000 x g for 10 min at 4°C
to completely remove platelets and precipitates.
4. Dilute samples according to the assay instruction
manual. dAliquot and store undiluted sample at –80°C
if not using immediately. Do not freeze dilutedsample.
Serum separating tubes (SST) are preferred
for superior separation quality.

Steps to a Successful Plasma Prep:
1. Centrifuge at 1,000 x g for 15 min at 4°C and transfer
into a clean tube.
2. R epeat centrifugation at 10,000 x g for 10 min at 4°C
to completely remove platelets and precipitates.
3. Dilute samples according to the assay instruction
manual.

Use collection tubes coated withanticoagulants (such as Na citrate or EDTA)
for better results.
Use protease inhibitor when profilingfor diabetes/metabolic markers.

Note: You may have to centrifuge plasma again at
3,000 x g for 5 min if the sample clotted duringextended storage. Care should be taken not to disturbpelleted material.

Steps to a Successful Cell Culture
Supernatant Prep:
1. Centrifuge at 1,000 x g for 15 min at 4°C.
2. S
 upplement with serum (5–10%) or BSA (0.5–1%)
to reconstitute the standard if using a serum-free
culture supernatant.
3. Typically, cell culture supernatant is analyzed
undiluted. However, if it is necessary to dilute thesample so that it is in the working range of thestandard curve, ensure the sample contains thesame amount of serum or BSA as the standard.
4. Aliquot and store at –80°C if not using sample
immediately.

Steps to a Successful Cell and Tissue
Homogenates Prep:
1. Rinse the tissue sample with cell wash buffer.*
2. If necessary, cut the tissue into small pieces (forexample, 3 x 3 mm) and transfer to a tissue grinder
on ice.
3. D
 ilute lysate at least twofold (1:2) in sample diluent** +0.5% BSA, to a final protein concentration of
200–900 µg/ml.
For analytes with high expression, a lysate
protein concentration as low as 50 µg/ml
may be sufficient.

Standards should be reconstituted in the same buffer
composition as your sample.

Dilute lysates as much as possible to reducethe detergent concentration. This will resultin optimum antibody binding during sample
incubation period.

* Available in Bio-Plex Pro™ cell signaling reagent kit (catalog #171-304006M).
** Available in Bio-Plex Pro reagent kit with flat bottom plate(catalog #171-304070M) or included in all preconfigured kits.

General Sample Tips
to MakeYour Life Easier!

Plan your experiment to ensure you use consumablesfrom a single lot.
Consistency is the key:
• Use low protein binding aliquot tubes to maintainconsistent assay performance• Dispense reagents using multichannel pipets• Ensure you use a lab timer to improve time uniformity
between incubation steps in the assay• Use dilution tubes to prepare serial dilution• Use reverse-pipetting technique to enhance liquid
dispensing accuracy• Incubate the reconstituted standards on ice for30 min• Resuspend beads thoroughly before removing
desired volume

Prepare 25% excess when possible.
Always run your standards, blanks, and samples
in duplicates or more.
Strepavidin-PE is light sensitive:• Cover the plate with foil during shaking steps• Store reagents protected from light
Use a 200–300 µl capacity pipet to remove beads fromthe stock tube to minimize volume loss. If necessary,
perform the volume transfer in two steps.
If not using immediately, then make sure
your sample is stored at –80°C in single-use
aliquots and minimize the number of freeze/thaw cycles.

For more assistance, visit our support website atwww.bio-rad.com/bio-plex
Follow us on@bioradlifesci

The Bio-Plex suspension array system includes fluorescently labeled microspheres andinstrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation.
xMAP is a trademark of Luminex Corporation.

www.bio-rad.com/bio-plex

Bio-RadLaboratories, Inc.
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