Bio-Rad Human MMP and TIMP Assays - Manual (Page 14)

12
Reconstitute Standards and Quality Controls
This procedure prepares enough standard to run each dilution in duplicate.
Note: The appearance of the lyophilized standards or control may vary
from a white pellet to clear crystals. Regardless of appearance, the vials
have passed QC specifications and perform accordingly.
1. Gently tap the vial containing the lyophilized standards on a solid
surface to ensure the pellet is at the bottom of the vial.
2. Reconstitute a single vial of standards with 781 µl of the appropriate
diluent.
Optional: at the same time, reconstitute the control vial with 250 µl of
the appropriate diluent as summarized in Table 4. The control does
not require further dilution.
3. Vortex the reconstituted standards and control at medium speed
for 5 sec, then incubate on ice for 30 min. It is important that
reconstitution of standards and control is started and ended at the
same time. Be consistent with this incubation time to ensure optimal
assay performance and reproducibility.
Prepare the Standard Dilution Series
The following procedure produces an eight-point standard curve with
a threefold dilution between each point. Pipet carefully using calibrated
pipets and use a new pipet tip for every volume transfer.
1. Label eight 1.5 ml polypropylene tubes S2 through S8 and Blank.
Alternatively, using Titertube ® micro test tubes may prove to be more
convenient if a multichannel pipet will be used to load the plate.
Table 4. Summary of recommended diluents for standards and controls.
Diluent for Standards
Sample Type and Controls* Add BSA
Serum and plasma Standard diluent HB None
Culture media, with serum Culture medium None
Culture media, serum-free Culture medium To 0.5% final**
Lavage, sputum, other fluids Sample diluent HB To 0.5% final**
* If using diluents other than standard diluent HB, users must establish their own control ranges.
** At least 0.5% final BSA is recommended to stabilize analytes and reduce adsorption to labware.