Background Cell migration is a vital process for growth and repair. behavior could be detected for both easy muscle mass cells (SMCs) and endothelial cells (ECs) when utilizing injury versus non-injury assays. SMCs migrated faster than HUVECs when stimulated by injury in the scrape wound assay, with rates of 1 1.26?% per hour and 1.59?% per hour on polystyrene and gelatin surfaces, respectively. The fastest overall migration took place with HUVECs on a gelatin-coated surface, with the in-growth assay, at a rate of 2.05?% per hour. The slowest migration occurred with the same AZD7762 kinase inhibitor conditions but on a polystyrene surface at a rate of 0.33?% per hour. Conclusion For SMCs, injury is usually a dominating AZD7762 kinase inhibitor factor in migration when compared to the two cell exclusion assays, regardless of the surface tested: polystyrene or gelatin. In contrast, the migrating surface, namely gelatin, was a dominating factor for HUVEC migration, providing an increase in cell migration over the polystyrene surface. Overall, the cell exclusion assays – the in-growth and out-growth assays, provide a means to determine real migratory behavior of cells in comparison to migration confounded by cell wounding and injury. Electronic supplementary material The online version of this article (doi:10.1186/s13036-015-0015-y) contains supplementary material, which is available to authorized users. to to mechanistic studies. Here we utilized three different migration assays to elucidate the contribution of different factors on cell migration, i.e. injury and surface. The cell exclusion assays (in-growth assay and out-growth assay) measure non-injury inward and outward migration, respectively. In contrast, the scrape wound assay steps inward cell migration after cell injury occurs. We hypothesized that the presence of injury and a biologically active surface, gelatin, would yield an increase in cell migration for both SMC and HUVEC. As expected for both cell types, the injury-inducing scrape wound assay provided the highest percent migration at 48?h, followed by the in-growth assay and then the out-growth assay. Additionally, SMCs experienced higher overall migration than HUVECs for all those three assays. We were successfully able to differentiate between wounding and non-wounding, with the difference best demonstrated with the non-injury out-growth assay. Lastly, the presence of a biologically active substrate (gelatin) increased HUVEC migration in all three assays. The gelatin surface provided multiple cell attachment sites that allowed cells to anchor and gain traction for subsequent cell migration. The utilization of these injury and noninjury, as well as inward vs. outward migration assays has allowed us to differentiate the different components of the migratory process (i.e. injury, surfaces) for a variety of cell types (i.e. SMC and HUVEC). Extension of our assay approaches to other cell types may show useful for controlling variables associated with cell migratory processes and in elucidating the relative contribution of these factors to the cell migration process. Methods Smooth muscle mass cell culture Main rat SMC cultures were established according to a modification of the method of Ross, AZD7762 kinase inhibitor et al. . Briefly, rat descending aorta was aseptically harvested, adherent excess fat and adventitia were removed and aortas were de-endothelialized via passage of an applicator. Aortic tissue was then minced and fragments were incubated (37?C, 5?% CO2) in Dulbeccos Modified Eagles Medium (DMEM, Life Technologies, Carlsbad, CA) for seven days to allow outgrowth. Main SMCs were cultured in T-75 tissue culture flasks (Thermo Scientific, Rochester, NY, USA) with supplemented DMEM. DMEM was supplemented with 10?% fetal calf serum (Life Technologies, Carlsbad, CA, USA), 1?% ( em v/v /em ) antibiotic-antimycotic (Life Technologies, Carlsbad, CA, USA), and 1?% ( em v/v /em ) 0.2?M?L-glutamine (Lonza Walkersville, Walkersville, MD, USA). Media was stored at 4?C for use up to 4?weeks. SMC were produced to 80?% or greater confluency and were passaged with trypsin-versene combination (Lonza Walkersville, Walkersville, MD, USA) before use in experiments. Only cells between AZD7762 kinase inhibitor passages 3 and 8 were used. HUVEC cell Rabbit Polyclonal to MLKL culture HUVECs were purchased from BD Biosciences (San Jose, CA, USA), and cultured on gelatin-coated T-75 tissue culture flasks with supplemented M199 medium (Life Technologies, Carlsbad, CA, USA). M199 was supplemented with 1?% ( em v/v /em ) 0.2?M?L-glutamine, 1.5?% ( em v/v /em ) 1?M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid from Lonza Walkersville, Walkersville, MD, USA), 1.8?% PSG (penicillin-streptomycin-glutamine from Lonza Walkersville, Walkersville, MD, USA), 15?% ( em v/v /em ) fetal.