MMP-9 expression is found at low levels in a majority of the cell lines. Yet, MMP9 expression is considerably stimulated when treated with phorbol myristate acetate (PMA) — a protein kinase C ligand.1
For analyzing MMP-9 in cell lines, the cells may have to be stimulated first with PMA to detect a signal.
Cell Stimulation with PMA
The below protocol has been improved for use with U937 cells.
- Cells should be cultured at 37 °C in RPMI 1640 medium that contains 10% fetal bovine serum (FBS) and 2 mM glutamine.
- As soon as the cell culture achieves a density of 0.5 – 1 x 106 cells/mL, 20 mL (2 x 107) cells should be transferred into a new plate or flask and then PMA should be added to bring it to a final concentration of 10 ng/mL.
- After incubation at 37 °C for 21 hours, Brefeldin A should be added to a final concentration of 3 µg/mL and incubation should be done for an additional 3 hours.
Note: Treatment with Brefeldin A inhibits protein secretion.
- Cells should be collected by centrifugation and then washed with phosphate-buffered saline, or PBS.
- The pellet should be frozen immediately with liquid nitrogen.
Note: Cells can be stored at −80 °C or instantly lysed for western blot analysis.
- To the frozen cell pellet, add 300 µL ice-cold lysis buffer and then resuspend the cells by gently pipetting.
Note: If cells stay clumped together, vortex the tube for 10 seconds, incubate the tube on ice for 3 minutes, and attempt to resuspend again.
- Incubate on ice for 10 minutes and then pipette the cells again to resuspend any residual pelleted material.
- Sonicate for three cycles of 40 seconds. Repeat sonication if the cell pellet is still visible.
Note: A slight yellow color indicates good lysis.
- To pellet the cell debris, centrifuge the lysate at 8000 rcf at 4 °C for 10 minutes.
- Finally, transfer the supernatant to a fresh tube, taking care not to transfer any of the cell debris.
Note: Lysate can either be stored at −80 °C or used instantly for western blotting.
Add proteinase inhibitor prior to use.
|NP-40 or Triton X-100
|SDS (sodium dodecyl sulfate)
|Tris, pH 8.0
- Roomi MW, Monterrey JC, Kalinovsky T, Rath M, Niedzwiecki A. Patterns of MMP-2 and MMP-9 expression in human cancer cell lines. Oncol Rep. 21, 1323–1333 (2009).
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