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Figure 2 | Journal of Neuroinflammation

Figure 2

From: Plasminogen activator inhibitor type 1 regulates microglial motility and phagocytic activity

Figure 2

Plasminogen activator inhibitor type 1 (PAI-1) promoted the migration of microglia in a concentration-dependent manner. (A) BV-2 microglial cells were seeded at a density of 8.0 × 104 cells/well in 96-well plates. When the BV-2 microglial cells had reached 80 to 90% confluence, a single scratch wound was made by using a 200 μl pipette tip, and the cell debris was removed by washing with PBS. Cells were treated with mouse PAI-1 protein (0 to 1000 ng/ml). At 0 and 24 hours, phase-contrast pictures of the wounds at three different locations were taken, and then the fold increase of migration distance was measured in three independent experiments. Results are given as mean ± SD (n = 3). *P < 0.05, NS = not significant, compared with the untreated control (lower panel). Representative images are shown (upper panel; original magnification,× 150). (B) The Boyden chamber assay was also performed to evaluate cell migration. BV-2 microglial cells (5 × 104 cells/upper well) placed in the Boyden chambers were exposed to mouse PAI-1 protein (0 to 1000 ng/ml), and then incubated at 37°C for 6 hours. Microglial cells that migrated through a membrane were stained and counted. Results are given as mean ± SD (n = 3). *P < 0.01; compared with the untreated control (lower panel). Representative images are also shown (upper panel; original magnification, × 100). (C) BV-2 microglial cells were treated with BSA (0 to 22 nmol/l) or PAI-1 (2.2 nmol/l; 100 ng/ml), followed by the wound-healing assay as described above. Results are given as mean ± SD (n = 3). *P < 0.05, NS = not significant, compared with the untreated control. (D) BV-2 microglial cells were seeded at the density of 5.0 × 103 cells/well in 96-well plate. Cells were treated with a mouse PAI-1 protein (100 ng/ml) and incubated at 37°C for 12–72 hours to evaluate cell proliferation. Proliferation curves are based on the 2,5-diphenyltetrazolium bromide (MTT) assay. Results represent the mean ± SD (n = 3). Proliferation of PAI-1-treated cells (open circle) was compared with the untreated control (filled square). (E) The Boyden chamber assay was performed to evaluate primary microglial cell migration. Primary microglia cultures (5 × 104 cells/upper well) placed in the Boyden chambers were exposed to mouse PAI-1 protein (100 ng/ml), and then incubated at 37°C for 24 hours. Microglial cells that migrated through a membrane were stained and counted. Results are mean ± SD (n = 3). *P < 0.05; compared with the untreated control (lower panel). Representative images are also shown (upper panel). (F) C6 glioma cells were seeded at the density of 8.0 × 104 cells/well in 96-well plate. Cells were treated with BSA (100 ng/ml) or PAI-1 (100 ng/ml), followed by the wound-healing assay as described above. Results are the mean ± SD (n = 3). *P < 0.05; compared with the untreated control.

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