In preparation for migration assays, two-well silicone culture inserts (from ibidi GmbH, Cat# 80209) were converted into one-well inserts by cutting with a sharp, sterile blade. Individual one-well inserts were placed in the middle of a 35 mm dish coated with Poly-Ornithine and Laminin. The boundary of the insert was marked on the back of each dish with a thin marker. For this assay, periventricular endothelial cells, control endothelial cells and endothelial cells derived without GABA and WNT7A were used at passage number P2 or P3, while GABA interneurons cultured for six weeks were used. 104 periventricular endothelial cells or endothelial cells derived without GABA and WNT7A were seeded in each insert in periventricular endothelial cell medium without GABA. Same number of GABAergic interneurons or control endothelial cells were seeded per insert in their respective manufacturer’s recommended medium. The insert was removed after 48 hours and cells cultured for five days. After 5 days, cells were fixed and fluorescently labeled with an anti-human CD31 antibody (for endothelial cells) or an anti-human β-Tubulin antibody (for neurons) and DAPI. The distance between each cell body and the edge of the insert- boundary was measured using ImageJ.
Two well silicone culture insert
Manufactured by Ibidi
Sourced in Germany
The Two-well silicone culture inserts are designed for cell culture applications. The inserts provide two separate growth compartments within a single culture vessel, allowing for the simultaneous culture of different cell types or experimental conditions.
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Lab products found in correlation
6 protocols using two well silicone culture insert
1
Endothelial Cell Migration Assay
2
SC Migration Evaluation in Gap-Closure Assay
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A gap-closure assay was performed to evaluate the efficacy of SC migration among groups using two-well silicone culture inserts (Ibidi GmbH, Martinsried, Germany) set with transparent dishes (Ibidi GmbH). The two-well inserts were first filled with 10% FBS DMEM and then seeded with SCs (1 × 104 cells/cm2) for 24 h to form a confluent monolayer, which was visible through the transparent dish. After the removal of the inserts, a 500 μm cell-free interval with clear demarcation was generated in the center of the dish. SCs were irrigated with PBS and then incubated in a serum-free medium at 37 °C. After replacing the serum-free medium with extracts of the control, PALD, and PALDE groups (in the same ratio as with the SC proliferation method), the migration of SCs from the medial edges of the interval across the 500 μm distance was observed for 48 h. Images were taken using light microscopy and digitalized (Leica QWin, Germany) after 0, 24, and 48 h of migration; data were quantified using ImageJ.
3
Migration Assay of Endothelial Cells and Neurons
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In preparation for migration assays, two-well silicone culture inserts (from ibidi GmbH, Cat# 80209) were converted into one-well inserts by cutting with a sharp, sterile blade. Individual one-well inserts were placed in the middle of a 35-mm dish coated with poly-ornithine and laminin. The boundary of the insert was marked on the back of each dish with a thin marker. For this assay, PVECs, control endothelial cells and endothelial cells derived without GABA and WNT7A were used at passage number P2 or P3, while GABA interneurons cultured for 6 weeks were used. In total, 104 PVECs or endothelial cells derived without GABA and WNT7A were seeded in each insert in PVEC medium without GABA. Same number of GABAergic interneurons or control endothelial cells were seeded per insert in their respective manufacturer’s recommended medium. The insert was removed after 48 h and cells cultured for 5 days. After 5 days, cells were fixed and fluorescently labeled with an anti-human CD31 antibody (for endothelial cells) or an anti-human β-tubulin antibody (for neurons) and DAPI. The distance between each cell body and the edge of the insert boundary was measured using ImageJ.
4
Tenocyte Migration Efficiency Assay
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The efficiency of tenocyte migration was determined through gap-closing assay by the use of two-well silicone culture inserts (Ibidi GmbH, Martinsried, Germany) inset on the transparent dishes (Ibidi GmbH, Martinsried, Germany) without coatings. Tenocytes were seeded in the culture inserts (8 × 103 cells/cm2) with 70 μL DMEM containing 20% FBS. After resting for 8 h, a single layer of confluent cells could be noted on the transparent dish. At this point, once the culture inserts were removed, a 500 μm gap was produced in the middle of the dish. Tenocytes were washed with PBS, and then incubated in a serum-free medium at 37 °C for 1 h. The medium was then replaced with EVs (100 μg/mL) diluted with culture media, and tenocytes were left to migrate for 24 h from the medial edges of the 500 μm gap. The migration assays were performed using the control group and EVs group. The migrated tenocytes were observed and captured under bright field microscopy with a camera (Leica QWin, Wetzlar, Germany) from 0 to 24 h. The percentage of remaining area was evaluated using Image J. All results were obtained in five independent experiments.
5
Cell Migration Assay with TGF-β
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For cell preparation, 3×104 cells were seeded into a two-well silicone culture insert (ib80209, Ibidi) in a 35 mm dish and cultured overnight. To stop cell proliferation, cells were treated with 10 µg/ml mitomycin C-containing medium for 2 h, and then the culture inserts were removed. Cells were washed twice with PBS and cultured in a medium with or without 10 ng/ml TGF-β for 24 h. Cell images were taken at 0 h, 12 h and 24 h time points.
6
Cell Migration Assay using Silicone Insert
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A two-well silicone culture insert (Ibidi, Germany) with a defined cell-free gap, suitable for migration assays, was used as a migration barrier. The two-well silicone culture insert was then placed in the middle of the plate. Subsequently, cells at a density of 5 × 105/ml were seeded onto the culture plate 70μL per well and allowed to proliferate to 100% confluence under standard laboratory conditions. After attachment, the silicone insert was removed, leaving a 500μm cell-free gap. Culture medium with different treatments was added respectively. Finally, an inverted microscope (Zeiss, Germany) was equipped to capture the migration distance of HUVECs to the cell-free zone during 12h. ImageJ software was used to measure the areas of the wound.
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