Bio-Rad Bio-Plex Pro™ TGF-β Assays - Manual (Page 18)

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Cell Culture Supernatant
1. Collect supernatants and perform centrifugation at 1,000 x g for 15 min
at 4°C. For cell lines cultured in serum-free culture media, collect samples
and add BSA as a carrier protein to a final concentration of at least
0.5% to stabilize protein analytes and to prevent adsorption to labware.
2. Transfer to a clean polypropylene tube. If cellular debris or precipitates
are present, centrifuge again at 10,000 x g for 10 min at 4°C.
3. Activate samples as described below and assay immediately or store
untreated/nonactivated samples in single-use aliquots at –70°C.
Avoid repeated freeze-thaw cycles.
Lavage, Sputum, and Other Biological Fluid Samples
Keep all samples on ice until ready for use. The appropriate sample
dilution factor should be determined by the user.
1. Centrifuge at 10,000 x g for 10 min at 4°C to clarify the sample.
2. Activate a portion of the sample as described below and, if needed,
dilute in Bio-Plex ® sample diluent with BSA added to a final
concentration of at least 0.5%.
Lysates
Users will need to optimize the lysis, sample dilution, and sample activation
methods to ensure that assay performance is fit for purpose.
1. Prepare the cell or tissue lysates according to the instructions provided
with the Bio-Plex cell lysis kit (catalog #171-304011 or 171-304012).
The protease inhibitors factor I and factor II are included in the kit. PMSF
needs to be added to lysis buffer at a final concentration of 2 mM. The
lysates should be free of particulate matter.
2. Determine the protein concentration of the lysate. It may be
necessary to test lyse your sample with different volumes of lysing
solution to obtain the specified protein concentration range.
3. Activate lysates as described under Sample Activation and Dilution.
Dilute the activated sample in sample diluent + 0.5% BSA. The
appropriate final lysate protein concentration should be determined
by the user. A starting range to try is 50 to 900 ug/ml.