Applying Phorbol Myristate Acetate to Stimulate MMP-9 Expression

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.

  1. Cells should be cultured at 37 °C in RPMI 1640 medium that contains 10% fetal bovine serum (FBS) and 2 mM glutamine.
  2. 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.
  3. 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.

  1. Cells should be collected by centrifugation and then washed with phosphate-buffered saline, or PBS.
  2. The pellet should be frozen immediately with liquid nitrogen.

Note: Cells can be stored at −80 °C or instantly lysed for western blot analysis.

Cell Lysis

  1. 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.

  1. Incubate on ice for 10 minutes and then pipette the cells again to resuspend any residual pelleted material.
  2. Sonicate for three cycles of 40 seconds. Repeat sonication if the cell pellet is still visible.

Note: A slight yellow color indicates good lysis.

  1. To pellet the cell debris, centrifuge the lysate at 8000 rcf at 4 °C for 10 minutes.
  2. 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.

Lysis Buffer

Add proteinase inhibitor prior to use.

. .
NaCl 150 mM
NP-40 or Triton X-100 1.0%
Sodium deoxychlorate 0.5%
SDS (sodium dodecyl sulfate) 0.1%
Tris, pH 8.0 50 mM

References

  • 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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Last updated: May 14, 2019 at 8:21 AM

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