Bio-Rad Human MMP and TIMP Assays - Manual (Page 19)

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 HB 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.
7. Prepare Coupled Beads
1. Use Tables 6–7 or the Calculation Worksheet on pages 35–36 to
calculate the volume of coupled beads and assay buffer needed to
prepare a 1x stock.
2. Add the required volume of Bio-Plex ® assay buffer to a 15 ml
polypropylene tube.
17
2. Add an adequate volume of cell lysis solution and grind the tissue
sample on ice using approximately 20 strokes.
Note: It may be necessary to lyse the samples with different volumes of
cell lysis solution to obtain the specified protein concentration range.
3. Transfer the ground tissue to a clean microcentrifuge tube and freeze
sample at –70°C. Freezing and thawing samples helps increase cell
lysis effects.
4. Thaw the sample and sonicate on ice (for example, with a Sonifier
450: Duty Cycle = 40, Output = 1, Pulse Sonicating = 18x).
5. Remove insoluble cellular matter as in Adherent Cells step 5 above.
6. Follow Adherent Cells steps 6–9 above.