Bio-Rad Bio-Plex Pro™ Human Th17 Cytokine Assays - Manual (Page 17)

Cell Culture Supernatant
1. Collect supernatants and centrifuge 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. We recommend testing undiluted samples first. If levels are
anticipated to be high, samples can be further diluted in culture
medium. Rarely would samples need to be diluted greater than 1:10.
4. Assay immediately or store 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 optimized by the user.
1. If required, dilute the sample in Bio-Plex sample diluent with BSA
added to a final concentration of 0.5%.
2. Centrifugation at 10,000 x g for 10 min at 4°C may be required to
clarify the sample.
Lysates
The Bio-Plex cell lysis kit is required for lysate preparation (available
separately, catalog #171-304011 and #171-304012). Refer to bulletin 5297
for a list of published articles on cytokine analysis in tissue samples.
1. Prepare the cell or tissue lysates according to the instructions provided
with the Bio-Plex cell lysis kit. The protease inhibitors, Factor I and 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.
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