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

4. Extremely lipemic samples may be filtered through a 0.2 µm filter to
prevent clogging.
5. Dilute samples with isotyping diluent following a 2-step dilution scheme
as noted below, vortexing for 5 sec between each dilution step.
1:40,000 dilution for IgG
1
, IgG
2
, IgG
3
, IgG
4
, IgA, IgM, and IgG total
Step 1. 5 µl of sample + 995 µl of diluent
Step 2. 5 µl of solution from first step + 995 µl of diluent
1:500 dilution for IgE
Step 1. 5 µl of sample + 120 µl of diluent
Step 2. 10 µl of solution from first step + 190 µl of diluent
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 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 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. Reconstitute and dilute the isotyping standard in the same medium
or matrix in which cells are prepared. Be sure to include all medium
components (such as FBS) as appropriate. To minimize error due
to lot-to-lot variation of culture media, use the same lot of culture
medium that was used to prepare the cells.
4. Assay immediately or store samples in single-use aliquots at –70°C.
Avoid repeated freeze-thaw cycles.
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