Bio-Rad Bio-Plex Pro TGF-β Assays User Manual

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

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system (30 min).

3. Prime wash station for flat bottom plate or set vacuum manifold to

–1 to –3" Hg for filter plate.

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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