Bio-Rad Bio-Plex Pro™ TGF-β Assays - Manual (Page 20)

18
Activation of Cell Culture Supernatant and Other
Biological Fluids
Samples may be run “neat” after activation/neutralization or be diluted as
required. The appropriate dilution factor should be optimized by the user.
Ensure a final sample volume after treatment and dilution of at least 125 µl
to allow for duplicate wells on the assay plate.
1. For example, if a 1:4 dilution is desired, activate the sample by
adding 10 µl acid to 50 µl sample. Mix thoroughly and incubate for
10 min at room temperature.
2. To neutralize sample, add 10 µl base. Mix thoroughly. Treated sample
volume is now 70 μl.
3. Dilute to 1:4 final in the same diluent used to prepare the standards.
In this example, starting sample volume was 50 μl and a 1:4 dilution
gives 200 μl. To reach a final volume of 200 μl, add 130 μl diluent to
70 μl treated sample
Note: Serum-containing culture medium may contain high concentrations
of TGF-b. A preliminary measurement of medium alone is recommended
to determine baseline levels.
6. Prepare Coupled Beads
Instructions are provided for diluting the coupled beads to a 1x concentration.
1. Use Tables 7–8 or the Calculation Worksheet on pages 37–38 to
calculate the volume of coupled beads and assay buffer needed.
2. Add the required volume of Bio-Plex assay buffer to a 15 ml
polypropylene tube.
3. Vortex the stock coupled beads at medium speed for 30 sec.
Carefully open the cap and pipet any liquid trapped in the cap back
into the tube. This is important to ensure maximum bead recovery.
Do not centrifuge the vial; doing so will cause the beads to pellet.