Cells were plated in the upper compartments of a Transwell tray (BD Biosciences) and allowed to migrate through the intervening membrane. Thereafter, cells were fixed in paraformaldehyde (3%), permeabilized in Triton X-100 (0.05%) and stained with crystal violet (0.02%). Cells on the upper side of the filter were removed and migrating cells were photographed. Invasion assays were performed using BioCoat Matrigel chambers. 3D spheroid cell invasion kits were from Trivigen. For trans-endothelial migration (TEM) assays, human umbilical vein endothelial cells (HUVEC) were grown to confluency on cell culture inserts. Tumor cells were added to the endothelial layer and left to transmigrate.
Transwell tray
Manufactured by BD
The Transwell tray is a laboratory equipment used for studying cell migration, permeability, and transport processes. It consists of a two-chambered system with a porous membrane that allows the movement of cells, molecules, or other substances between the upper and lower compartments.
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4 protocols using transwell tray
1
Cell Migration and Invasion Assays
2
Transwell-based Cell Migration and Invasion Assay
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Cells (105 cells/insert) were plated in the upper compartment of a Transwell tray (BD Bioscience, San Jose, California). The lower compartment was coated with 25 ug/ml collagen for 2 h prior to cell plating. Cells were allowed to migrate through the intervening nitrocellulose membrane for 20 hr in the presence of growth factor in the lower chamber. Thereafter, cells were fixed in 3% PFA in PBS, permeabilized in Triton X-100 (0.05%) and stained with Methyl Violet (0.02%). Non-migrating cells, growing on the upper side of the filter, were removed with a cotton swab and cells that had migrated photographed. For cell invasion assays, 105 cells/insert were plated in the upper compartment of BioCoat Matrigel Chambers (BD Bioscience, San Jose, California), were allowed to invade through matrigel for 48 hr and processed as above.
3
Cell Migration and Invasion Assay
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To examine cell migration, cells (U2OS 4 × 104, 769-P 3 × 104, HeLa and MDA-MB-231 8 × 104 cells per well) were plated in the upper compartment of a 24-well Transwell tray (pore size 8 μm, BD Biosciences) in full medium lacking FBS. Medium supplemented with 10% FBS was used as a chemoattractant in the lower chamber. Cells were allowed to migrate through the intervening nitrocellulose membrane (8 μm pore size) during 12 h of incubation at 37 °C. For cell invasion examination, 4 × 104 U2OS cells per well were plated in the upper compartment of a 24-well Transwell tray pre-coated with Matrigel (pore size 8 μm, Corning, 354480) instead, and cells were incubated at 37 °C for 18 h. Cells on the upper surface of the insert were then removed by a cotton swab. Cells migrated to the lower surface of the insert were fixed with methanol, stained with crystal violet, and then photographed. The numbers of migrated cells in five random fields under the microscope were counted for the quantitative analysis. Assays were repeated at least three times. Data were shown as mean ± SD and p-value was calculated by Student’s t-test.
4
Assessing Cell Proliferation, Migration, and Invasion
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Proliferation of cells was assessed using MTT or XTT. Brie y, cells were seeded in triplicates in 96-well plates and treated for the indicated time intervals. Subsequently, MTT was added and following 3 hours at 37°C, we dissolved in DMSO the water insoluble formazan crystals, which form in metabolically active cells. Optical density was measured at 570 nm. The XTT-based assays followed the manufacturer instructions.
Cell migration, invasion, and 3D assays Cells were plated in the upper compartments of a Transwell tray (BD Biosciences) and allowed to migrate through the intervening membrane. Thereafter, cells were xed in paraformaldehyde (3%), permeabilized in Triton X-100 (0.05%) and stained with crystal violet (0.02%). Cells on the upper side of the lter were removed and migrating cells were photographed. Invasion assays were performed using BioCoat Matrigel chambers. 3D spheroid cell invasion kits were from Trivigen. For trans-endothelial migration (TEM) assays, human umbilical vein endothelial cells (HUVEC) were grown to con uency on cell culture inserts, tumor cells were added to the endothelial layer and left to transmigrate.
Cell migration, invasion, and 3D assays Cells were plated in the upper compartments of a Transwell tray (BD Biosciences) and allowed to migrate through the intervening membrane. Thereafter, cells were xed in paraformaldehyde (3%), permeabilized in Triton X-100 (0.05%) and stained with crystal violet (0.02%). Cells on the upper side of the lter were removed and migrating cells were photographed. Invasion assays were performed using BioCoat Matrigel chambers. 3D spheroid cell invasion kits were from Trivigen. For trans-endothelial migration (TEM) assays, human umbilical vein endothelial cells (HUVEC) were grown to con uency on cell culture inserts, tumor cells were added to the endothelial layer and left to transmigrate.
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