To evaluate the influence of TNF-α-induced MMP-9 expression on the metastatic activity of GES-1 cells, an in vitro Transwell assay (Becton-Dickinson, Franklin Lakes, NJ, United States) was used. Cells at a concentration of 1 × 105 cells/100 mL in serum-free RPMI 1640 medium were added to the upper chamber, and the Matrigel-uncoated insert was inserted in the lower chamber, which contained 20% FBS + RPMI 1640 medium. The migration assay was run for 24 h following a previously described standard protocol[39 (link),42 (link)].
Transwell assay
Manufactured by BD
Sourced in United States
The Transwell assay is a cell culture system that allows for the study of cell migration and invasion. It consists of a two-chamber setup, with the upper chamber containing cells and the lower chamber containing a chemoattractant or other stimuli. The cells are able to migrate through a porous membrane separating the two chambers, and their movement can be quantified.
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Lab products found in correlation
Matrigel, by BD (9 mentions)
Transwell plate, by BD (2 mentions)
Matrigel, by Corning (2 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Matrix glue, by BD (1 mentions)
6 well plate, by Corning (1 mentions)
Matrigel solution, by BD (1 mentions)
Dm6000b, by Leica (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Diff quick staining kit, by Sysmex (1 mentions)
Matrigel pre coating, by BD (1 mentions)
Phase contrast light microscope, by Olympus (1 mentions)
Eclipse ti u, by Nikon (1 mentions)
Optical microscope, by Leica (1 mentions)
Transwell invasion assay, by BD (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Turbofect reagent, by Thermo Fisher Scientific (1 mentions)
37 protocols using transwell assay
1
Evaluating TNF-α-induced MMP-9 and Metastasis
2
Transwell Assay for Cervical Cancer Migration
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To evaluate the migration capability of cervical carcinoma, a Transwell assay (Becton Dickinson, San Diego, CA, USA) was used according to the manufacturer’s protocol. Briefly, the upper chamber was coated with 100 μL Matrigel (Thermo Fisher Scientific) at 37°C for 30 minutes. After that, lentivirus-transduced Ca-Ski and HeLa cells were resuspended and plated in the upper chamber in 200 μL DMEM without FBS. In the lower chamber, 500 μL DMEM +10% FBS was added to act as chemoattractant. After 24 hours, the upper chamber and non-migrated cells were removed, and the lower chamber was fixed with ethanol and stained with crystal violet. The transwell was then placed on an Olympus IX-70 inverted microscope fluorescent microscope (Olympus Corporation, Tokyo, Japan). Relative migration capability was measured as the number of migrated cells for each experimental condition, and then normalized to control.
3
Assessing Metastatic Potential via Transwell Invasion Assay
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To assess the influence of DEK overexpression or depletion in GC cell lines on metastatic activity, the in vitro Transwell assay (Becton-Dickinson, Franklin Lakes, NJ, USA) was employed. After adjusting the density to 1 × 105 cells/100 μL serum-free RPMI, cells were added to the upper chamber and a Matrigel-coated (invasion assay) insert used to assess invasive capability, as described previously [46 (link)].
4
Transwell Cell Migration Assay
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Cell migration assay was performed by using the in vitro Transwell assay (Becton-Dickinson, Franklin Lakes, NJ, USA) with 8 µm pore membrane insert. Cell number was adjusted to 5 ×105 cells/ml in 100 μl suspension with serum-free DMEM, and the cells were seeded on upper chambers of the Transwell plate (Becton-Dickinson). The medium in the lower chamber was DMEM supplemented with 20% FBS. The cells were incubated for 16-20 h at 37 °C and allowed to migrate to the lower chamber. Migratory cells were detected via crystal violet staining and counted using Image J software.
5
Transwell Assay for Cell Migration and Invasion
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The migration and invasion abilities of C33A cells were detected using the Transwell assay with Transwell plates (BD Falcon, Bedford, MA, USA) [22 (link)]. For the invasion assay, Matrigel (Corning, Corning, NY, USA) was loaded into the upper chambers of 24-well Transwell inserts with 8 mm pore polycarbonate filters. The 24-well Transwell chambers without Matrigel were used for migration assays. C33A cells (5 × 104) were seeded into the upper chambers containing DMEM media and 1% FBS. DMEM media containing 10% FBS was added into the bottom chambers. After 48 hours of culture, the invading and migrating cells on the membrane bottom were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Images of the invading and migrating cells were observed under a light microscope (Olympus Optical Co., Ltd., Tokyo, Japan) with five random fields.
6
Silencing PDHA1 Impacts BE(2)-C Cell Viability, Migration, and Invasion
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BE(2)-C cells were transfected with PDHA1 siRNAs (Tsingke, China) for loss-of-function experiments (The sequences used for PDHA1 silencing are shown in Supplementary Table 5B
7
Transwell Assay for Cell Migration and Invasion
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Migration and invasion assays were determined using a BD Transwell assay (pore size 8 µm) with or without Matrigel (BD Biosciences, Franklin Lakes, NJ, USA), in accordance with the manufacturer's protocol. Following transfection, Saos-2 or U-2OS cells (5×104) were seeded into the upper wells on a filter coated with or without Matrigel in DMEM without serum, and the lower chambers were filled with DMEM supplemented with 10% FBS, which acted as a chemoattractant. The cells were cultured for 24 h at 37°C. Subsequently, the non-migrated or invaded cells on the upper surface of the filter were removed using cotton swabs. Cells that had migrated or invaded into the bottom surface were fixed with 75% ethanol and stained with 1% crystal violet solution at room temperature for 20 min. The relative degree of cell migration or invasion was quantified using a light microscope (magnification, ×100) and normalized to the control cells in 5 fields of view.
8
Transwell Assay for Cell Invasion and Migration
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BD Transwell assay (BD Biosciences, Franklin Lakes, NJ, USA) was used to determine the effects of curcumin or its analogs on PC3 or PLS10 cell invasion and migration. Polyvinylpyrrolidone‐free polycarbonate filters (8 μmol/L pore size) were coated with 10 μg/mL fibronectin (migration assay) along with 15 μg matrigel per filter (invasion assay). Fibronectin (10 μg/mL) in 0.1% FBS RPMI‐1640 or 10% FBS RPMI‐1640 was added to the lower chamber as a chemoattractant for PC3 cells or PLS10 cells, respectively. PC3 cells or PLS10 cells (2.0 × 106 cells/well) were plated in the upper chamber with or without curcumin or its analogs at a non‐toxic dose (IC20) and incubated for 24 or 48 hours for PC3 cells or PLS10 cells, respectively. Invading or migrating cells were fixed with methanol for 5 minutes and then stained with 0.5% crystal violet in 20% methanol for 30 minutes. Percentages of areas occupied with cells were then determined with IMAGE J 1.410 (National Institute of Mental Health, Bethesda, MD, USA).
9
Cell Invasion and Migration Assay
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Cell invasion and migration ability were detected by BD Transwell assay according to the previous published method.19 (link)
10
Transwell Assay for Invasion Analysis
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The influence of LINC01013 on invasion ability in vitro was determined by Transwell assay (Falcon BD, Franklin Lakes, NJ) using LINC01013-depleted KARPAS-299 or LINC01013-overexpressed SR-786 cells, as described previously15 (link). Briefly, cell density was adjusted to 105 cells/ml, and 100 μl of the suspension was seeded into upper chambers of the Transwell plate, either coated (invasion) or not coated (migration) with Matrigel (Becton-Dickinson). For both assays, the pore size of the upper chamber was 8 mm. The medium in the upper chamber was serum-free DMEM, and the lower chamber contained DMEM supplemented with 20% FBS, included as a chemoattractant. After incubation for 24 h at 37 °C, cells traversing the filter from the upper to lower chamber were stained with crystal violet and counted. Experiments were repeated at least three times.
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