Bio-Rad Human MMP and TIMP Assays - Manual (Page 16)

14
Serum and Plasma
Note: If using plasma, EDTA or citrate is preferred as an anticoagulant.
Heparin-treated plasma, while compatible with Bio-Plex Pro
™ assays,
may absorb certain soluble proteins of interest. Avoid using hemolyzed
samples, as this may lead to false positive results.
1. Draw whole blood into collection tubes containing anticoagulant.
Invert tubes several times to mix.
2. For serum, allow blood to clot at room temperature for 30 to 45 min.
For plasma, proceed directly to the centrifugation steps.
3. Perform centrifugation at 1,000 x g for 15 min at 4°C and transfer the
serum or plasma to a clean polypropylene tube.
4. To completely remove platelets and precipitates, centrifuge again at
10,000 x g for 10 min at 4°C. Alternatively, filter the samples with a
0.8/0.2 μm dual filter to prevent clogging.
5. Dilute samples four–fold (1:4) by adding 1 volume of sample to
3 volumes of Bio-Plex ® sample diluent HB (for example, 50 µl
sample + 150 µl sample diluent HB).
6. Assay samples immediately or aliquot into single-use tubes and store
at –70°C. Avoid repeated freeze-thaw cycles.
Cell Culture Supernatant
1. Collect supernatants by pelleting cells 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.
Table 5. Summary of recommended sample diluents and dilution factors.
Sample Type
Dilution Factor Diluent
Serum and plasma 1:4 dilution Sample diluent HB
Fluids 1:4 dilution Diluent + 0.5% BSA w/v