Mean area of gelatin degradation was quantified by counting a degraded area in 15–20 different fields containing approximately the same number of cell nuclei. For Matrigel migration assays, BMDMs were detached and starved in DMEM without serum for a total of 3 h. After 2 h of starving, the cells were labeled with the fluorescent dye Celltracker Blue CMAC (Invitrogen) according to the producer instruction. A total of 105 cells in DMEM without serum were then plated on BioCoat Matrigel Invasion Chambers (BD Biosciences) for 24 h. Nonmigrated cells were removed and migrated cells were counted by reading the fluorescence on the bottom side of the inserts with a Victor Multilabel

Plate Reader (PerkinElmer). For trans-endothelial migration assays, H5V cells, an epithelial cell line kindly provided by E. Dejana (FIRC Institute Proteasome inhibitor of Molecular Oncology, Milan, Italy) were plated on FluoroBlok Inserts (Falcon) for 3 days until they formed a confluent monolayer, and then activated with 5 ng/mL TNF for 2 h in DMEM. find more A total of 105 BMDMs labeled with Celltracker Blue CMAC (Invitrogen) as above described for Matrigel assays, and resuspended in DMEM without serum, were then plated on FluoroBlok inserts coated with TNF-activated H5V cells for 22 h. Percentage of migrated cells was calculated by reading the fluorescence

with a Victor Multilabel Plate Reader (PerkinElmer). Cell migration in 2D was assessed by scraping a confluent monolayer of BMDMs with a pipette tip. Then the number of cells migrating into the open space was assessed microscopically [[12]]. Quantification of migrated cells was performed counting cells migrated into the wound in ten different fields. Cells were lysed with sample buffer: 25 mM Tris, pH 6.8, 50 mM β-mercaptoethanol, 1% SDS and 5% glycerol and then analyzed with Odyssey Infrared Imaging Astemizole System (Li-cor Biosciences, Nebraska, USA) using specific antibodies. The Student’s t-test has been applied to examine the statistical significance of differences between the data. Values of *p < 0.05 or **p < 0.01, ***p

< 0.001 were taken as significant. This work was supported by a grant from Italian Association for Cancer Research (AIRC) to GB (grant 2010). The authors are indebted to Clifford A Lowell (UCSF) for having made available to them mice with the genetic deficiency of Hck and/or Fgr generated in his laboratory. The authors declare no financial or commercial conflict of interest. Disclaimer: Supplementary materials have been peer-reviewed but not copyedited. "
“Macrophages (Mϕ) are professional antigen-presenting cells, but when they accumulate at sites of inflammation, they can inhibit T-cell proliferation. In experimental autoimmune uveoretinitis, this limits the expansion of T cells within the target organ.