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

Yusuke Shiode, Yuki Morizane, Ryo Matoba, Masayuki Hirano, Shinichiro Doi, Shinji Toshima, Ryoichi Araki, Mika Hosogi, Kosuke Takahashi, Yuki Kanzaki, Tomoko Yonezawa, Fumio Shiraga

Research output: Contribution to journalArticle

Abstract

Objective To examine the usefulness of room temperature vulcanizing (RTV) silicone rubber as a barrier material for cell exclusion zone assays. Methods We created barriers using three types of RTV silicone rubber 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 fibronectin by 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 fibronectin on MIO-M1 cell migration and the effect of migration (with fibronectin coating) on basic fibroblast growth factor (bFGF) expression in MIO-M1 cells. Results Of the three types of RTV silicon rubber 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 fibronectin that 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. Fibronectin significantly 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 silicone rubber 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.

Original languageEnglish
Article numbere0190198
JournalPLoS One
Volume12
Issue number12
DOIs
Publication statusPublished - Dec 1 2017

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Silicone Elastomers
silicone
rubber
Fibronectins
Assays
ambient temperature
fibronectins
Temperature
assays
Polymerase chain reaction
Rubber
Fibroblast Growth Factor 2
cells
Transcription
cell movement
Cell Movement
fibroblast growth factor 2
Extracellular Matrix Proteins
Reverse Transcription
Silicon

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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A novel cell exclusion zone assay with a barrier made from room temperature vulcanizing silicone rubber. / Shiode, Yusuke; Morizane, Yuki; Matoba, Ryo; Hirano, Masayuki; Doi, Shinichiro; Toshima, Shinji; Araki, Ryoichi; Hosogi, Mika; Takahashi, Kosuke; Kanzaki, Yuki; Yonezawa, Tomoko; Shiraga, Fumio.

In: PLoS One, Vol. 12, No. 12, e0190198, 01.12.2017.

Research output: Contribution to journalArticle

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abstract = "Objective To examine the usefulness of room temperature vulcanizing (RTV) silicone rubber as a barrier material for cell exclusion zone assays. Methods We created barriers using three types of RTV silicone rubber 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 fibronectin by 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 fibronectin on MIO-M1 cell migration and the effect of migration (with fibronectin coating) on basic fibroblast growth factor (bFGF) expression in MIO-M1 cells. Results Of the three types of RTV silicon rubber 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 fibronectin that 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. Fibronectin significantly 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 silicone rubber 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.",
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AU - Matoba, Ryo

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AU - Doi, Shinichiro

AU - Toshima, Shinji

AU - Araki, Ryoichi

AU - Hosogi, Mika

AU - Takahashi, Kosuke

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AU - Yonezawa, Tomoko

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