Bio-Rad Human MMP and TIMP Assays - Manual (Page 17)

3. We recommend testing undiluted samples first. If high levels of
analyte are expected, 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.
Cell Lysates
The Bio-Plex Pro ™ cell signaling reagent kit (catalog #171-304006M) is
required for preparing lysates derived from cell culture and tissue samples.
Just before use, prepare an adequate volume of cell lysis buffer by adding
PMSF and cell lysis factor QG.
n
Prepare 500 mM PMSF by dissolving 0.436 g PMSF in 5 ml DMSO.
(Store as aliquots at –20°C). Add PMSF to the cell lysis buffer at a final
concentration of 2 mM
n
Reconstitute cell lysis factor QG to 100x with 250 μl of diH
2
O and vortex
to mix. Add the reconstituted factor to the cell lysis buffer to a final 1x
working concentration
Adherent Cells
1. Stop the treatment reaction by aspirating the culture medium and
quickly rinsing the cells with ice-cold cell signaling cell wash buffer
(bottle with the blue cap). The volume of buffer required is the same
as the volume of aspirated cell culture medium. Keep the cells on ice
during all steps when possible.
2. Completely remove the buffer before lysing the cells.
3. Immediately add the cell lysis buffer to the cells. The amount of lysing
solution needed depends on the cell density in the culture vessel (for
example, add 1.5–2 ml of lysis buffer to a 10 cm dish that is ~80%
confluent).
Note: It may be necessary to lyse the samples with different volumes of cell
lysis solution to obtain the specified protein concentration range.
4. Scrape the cells with a cell scraper, collect cell suspension into an
appropriately sized tube and gently rock for 20 min at 4°C.
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