A novel cell exclusion zone assay with a barrier made from room temperature vulcanizing silicone rubber

Objective To examine the usefulness of room temperature vulcanizing ( RTV) silicone rubberas a barrier material for cell exclusion zone assays. Methods We created barriers using three types of RTV siliconerubber with differing viscosities. We then assessed the adherence of these barriers to culture dishes and their ease of removal from the dishes. We tested the effect of the newly created barriers on the extracellular matrix (ECM) protein fibronectinby attaching and then removing them from fibronectin-coated culture dishes. We also conducted cell exclusion zone assays with MIO-M1 cells using this new barrier in order to measure cell migration. We used real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining to measure the effect of fibronectinon MIO-M1 cell migrationand the effect of migration (with fibronectincoating) on basic fibroblast growth factor (bFGF) expression in MIO-M1 cells. Results Of the three types of RTV siliconrubber tested, KE-3495-T was the best in terms of adherence to the dish and ease of removal from the dish. When barrier attachment and removal tests were performed, this rubber type did not have an effect on the fibronectinthat coated the dish. In the cell exclusion assay, removal of the barrier revealed that a cell-free area with a distinct margin had been created, which allowed us to conduct a quantitative assessment of migration. Fibronectinsignificantly promoted the migration of MIO-M1 cells (P = 0.02). In addition, both real time RT-PCR and immunohistological staining indicated that bFGF expression in migrating MIO-M1 cells was significantly higher than that in non-migrating cells (P = 0.03). Conclusions RTV siliconerubber can be used to create an effective barrier in cell exclusion zone assays and allows simple and low-cost multi-parametric analysis of cell migration.