Bio-Rad Bio-Plex Pro™ TGF-β Assays - Manual (Page 19)

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Note: For optimal assay performance, it is important to dilute lysates
as much as possible to reduce the detergent concentration.
4. Store untreated/nonactivated lysates at –20°C to –70°C. Avoid
multiple freeze-thaw cycles
Sample Activation and Dilution
First, prepare samples as described above. To measure immunoreactive
TGF-b, it is necessary to treat all sample types with the following activation
procedure. Samples should be assayed immediately after the neutralization
step. Do not activate the TGF-b standards.
Activation of Serum and Plasma
1.
To activate the sample, add 1 volume of acid (1 N HCl) to 5 volumes
of sample. For example, add 5 μl acid to 25 μl of sample. Mix
thoroughly and incubate for 10 min at room temperature.
2. To neutralize the sample, add a volume of base (1.2 N NaOH/0.5 M
HEPES) equal to the volume of 1 N HCl used. In this example, add
5 μl base and mix thoroughly. Treated sample volume is now 35 μl.
3. The recommended dilution is 1:16 of the starting (untreated) sample
volume. In this example, starting sample volume was 25 μl and a 1:16
dilution gives 400 μl. To reach a final volume of 400 µl, add 365 μl
Bio-Plex sample diluent to 35 μl treated sample.
Note: To achieve neutral pH of a sample (pH 7.2 to 7.6), the actual volume
of base required may vary depending on initial sample pH and the buffering
capacity of the sample. Verify pH using pH paper before running the assay.
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.
** At least 0.5% final w/v BSA is recommended to stabilize analytes and reduce adsorption to
labware.
Table 6. Summary of recommended sample diluents and dilution factors.