Bio-Plex Pro™ Assays
Quick Guide 5
For use with Instruction Manual #
TGF-b Assays 10024984
This guide can be used to prepare and run a full 1 x 96-well assay plate. For
more information on a given step, refer to the complete instruction manual.
New users can download the manual at www.bio-rad.com/bio-plex.
Important: Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from the protocol may result in low assay signal and assay variability.
Initial Preparation
1. Plan the plate layout.
2. Start up/warm up the Bio-Plex
n
Bring assay buffer, wash buffer, and sample diluent to room
temperature (RT). Keep other items on ice until needed
n
Begin to thaw frozen samples
®
system (30 min).
3. Prime wash station for flat bottom plate or set vacuum manifold to
–1 to –3" Hg for filter plate.
™
4. Follow the prompts in Bio-Plex Manager
software to calibrate the
system. This can be done now or during an incubation step.
5. Mix 1 volume of Bio-Plex standard diluent with 3 volumes of Bio-Plex
sample diluent (each supplied in the kit). The resulting solution is used
for reconstitution and subsequent dilution of standards. This results in a
serum-matrix based diluent that mimics the matrix in 1:16 diluted serum
and plasma samples. For samples in serum-free media and other biological
fluids, use a diluent that most closely matches the sample matrix. Add carrier
protein such as BSA at a final concentration of 0.5% (w/v).
Bio-Plex Pro Assay Quick Guide 5
6. Reconstitute a single vial of standards in 500 µl of a diluent similar to the
final sample type or matrix as shown below. Vortex for 5 sec and incubate
on ice for 30 min.
Samp le Type Dilue nt for St andard Add BSA
Serum and plasma Standard/sample diluent mix (1:3) None
Culture media, with serum Culture media None
Culture media, serum-free Culture media To 0.5% final (w/v)
Lavage, lysate, other fluids Sample diluent To 0.5% final (w/v)
7. Prepare a fourfold standard dilution series and blank as shown below.
Vortex for 5 sec between liquid transfers.
128 50 50 50 50 50 50 50
Transfer Volume, µl
Reconstituted
Standard
72 150 150 150 150 150 150 150 150
S1 S2 S3 S4 S5 S6 S7 S8 Blank
Diluent, µl
8. After thawing samples, activate by adding 1 volume of 1 N HCI to
5 volumes of sample. Vortex, incubate at RT for 10 min. Neutralize by
adding the same volume of base (1.2 N NaOH/0.5 M HEPES). After
treatment, dilute samples as shown below.
Sample Type Diluent Add BSA Sample Dilution
Serum and plasma Sample diluent None 1:16 final*
Culture media, with serum Culture media None User optimized
Culture media, serum-free Culture media To 0.5% final User optimized
Lavage, lysate, other fluids Sample diluent To 0.5% final User optimized
* For example, activate 25 μl sample, neutralize, and bring to a final volume of 400 μl.
9. Vortex the 20x coupled beads for 30 sec and dilute to 1x in Bio-Plex
assay buffer as shown below. Protect from light.
# of Wells 20x Beads, µl Assay Buffer, µl Total Volume, µl
96 288 5,472 5,760
Bio-Plex Pro Assay Quick Guide 5
Running the Assay
Note:
Make sure all assay components are at RT before proceeding.
1. Prewet filter plate with 100 µl Bio-Plex assay buffer (skip for flat bottom).
2. Vortex the diluted (1x) beads. Add 50 µl to each well of the assay plate.
3. Wash two times with 100 µl Bio-Plex wash buffer.
4. Vortex samples, standards, blank. Add 50 µl to each well.
5. Cover plate with sealing tape and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 2 hr at RT.
Note:
850 rpm provides equivalent performance to recommended shaker
settings in previous manuals (1,100 rpm for 30 sec, 300 rpm for incubation).
6. With 10 min left in the incubation, vortex the 20x detection antibodies
for 5 sec and quick-spin to collect liquid. Dilute to 1x in detection
antibody diluent as shown below.
# of Wells 20x Detection Ab, µl Detection Ab Diluent, µl Total Volume, µl
96 150 2,850 3,000
7. Wash the plate three times with 100 µl wash buffer.
8. Vortex the diluted (1x) detection antibodies. Add 25 µl to each well.
9. Cover and incubate at 850 ± 50 rpm, as in Step 5, for 1 hr at RT.
Meanwhile, prepare Bio-Plex Manager software protocol; enter
standard S1 values provided in the assay kit.
10. With 10 min left in the incubation, vortex the 100x SA-PE for 5 sec, quick-
spin to collect liquid, and dilute to 1x as shown below. Protect from light.
# of Wells 100x SA-PE, µl Assay Buffer, µl Total Volume, µl
96 60 5,940 6,000
11. Wash the plate three times with 100 µl wash buffer.
12. Vortex the diluted (1x) SA-PE. Add 50 µl to each well.
Bio-Plex Pro Assay Quick Guide 5
13. Cover and incubate at 850 ± 50 rpm, as in Step 5, for 30 min at RT.
14. Wash the plate three times with 100 µl wash buffer.
15. Resuspend beads in 125 µl assay buffer. Cover and shake at
850 ± 50 rpm, as in Step 5, for 30 sec.
16. Remove the sealing tape and read plate using the settings below.
Instrument RP1 (PMT) DD Gates Bead Events
Bio-Plex 100, 200* Low 5,000 (low), 25,000 (high) 50
Bio-Plex 3D* Standard Select MagPlex beads 50
®
Bio-Plex
* Or similar Luminex-based system.
MagPlex is a trademark of Luminex Corporation.
The Bio-Plex suspension array system includes fluorescently labeled microspheres and
instrumentation licensed to Bio-Rad Laboratories, Inc. by the Luminex Corporation.
MAGPIX
™
*
N/A, use default instrument settings Default
Life Science
Group
Bio-Rad
Laboratories, Inc.
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