The cell migration assay was carried out using a transwell system with pore size of 8 µm (Corning Costar, Corning, NY, USA). The cells (0.8 × 105 cells per well) were seeded into the upper chamber in serum free DMEM, while conditioned medium from NIH-3T3 was applied to the lower chamber. After 4 h incubation, migrated cells on the bottom surface of the membrane were fixed with 1% PFA, non-migrated cells on the top of the membrane were gently removed by cotton bud while the migrated cells at the bottom of the membrane were stained with DAPI. Migrated cells were counted in five random microscopic fields (original magnification, 10×). The cell invasion assay was carried out using a transwell system with pore size of 8 µm (Corning Costar, USA), coated with Matrigel (BD Biosciences, San Jose, CA, USA). Similarly, the cells (1 × 105 cells per well) were seeded into the upper chamber in DMEM supplemented with 1% FBS, while medium with 10% FBS was applied to the lower chamber. After 24 h, invading cells were quantified as described above.
Transwell system
Manufactured by Corning
Sourced in United States, China
The Transwell system is a cell culture insert that allows for the study of cells in a layered environment. It consists of a porous membrane that separates the upper and lower chambers, enabling the analysis of cell migration, permeability, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (75 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (13 mentions)
Crystal violet, by Merck Group (11 mentions)
Matrigel, by Corning (9 mentions)
Crystal violet, by Beyotime (9 mentions)
24 well plate, by Corning (7 mentions)
Matrigel, by Merck Group (7 mentions)
Lipopolysaccharides (lps), by Merck Group (6 mentions)
Inverted microscope, by Olympus (5 mentions)
Polycarbonate membrane, by Corning (4 mentions)
Trizol, by Merck Group (4 mentions)
Biocoat invasion system, by BD (4 mentions)
Tryple express, by Thermo Fisher Scientific (4 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (4 mentions)
Matrigel coated membrane, by BD (4 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Polyester pet membrane, by Corning (3 mentions)
548 protocols using transwell system
1
Cell Migration and Invasion Assay
2
Transwell Assay for Monocyte-Derived Langerhans Cell Migration
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Migration of mo-LCs was measured in triplicate with a transwell system (cat. no.3421, 24-well plates; 5.0 µm pore size; Corning Costar, NY). RPMI 1640 with 1% FCS alone, with varying doses of M-CSF or IL34 (cat. no. 200-34, PeproTech) added to the lower chamber. Wells with medium alone were used as a control for spontaneous migration. A total of 2.5 × 10 5 cells in 200 µl was added to the upper chamber and incubated at 37°C for 4 h. In some migration experiments, cells were pretreated with GW2580 (1uM, cat. no. SML1047, Sigma-Aldrich) or with BLZ945 (0.5 M, Selleckchem) for 60 min at 37°C. For migration of BDCA1 + LC, 1.5 × 10 5 cells in 200 µl were added to the upper chamber of a transwell system (5.0 µm pore size; Corning Costar) and harvested after 6 h of migration towards M-CSF (50 ng/ml). Mo-LCs and BDCA1 + LCs were not enriched for CD207. Cells migrated into the lower chamber were harvested, concentrated to a volume of 300 µl of PBS, and counted by flow cytometry. Events were acquired for a fixed time of 60 s. The counts fell within a linear range of the control titration curves obtained by testing increasing cell concentrations. Values are given as the mean number of migrated cells ± SEM.
3
Coculturing Tumor, NK, and T Cells
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Tumor cells were plated at a density of 2,000 or 4,000 cells/well in either 96-well flat-bottomed plates or the lower chamber of a transwell system (Corning, NY, USA) in DMEM supplemented with 10% FBS. MSCs were plated at a density of 2,000 or 40,000 cells/well in either 96-well flat-bottomed plates or the upper chamber of a transwell system (Corning, NY, USA) in DMEM supplemented with 10% FBS. The following day, natural killer (NK) or T cells were added to the cultures on top of the tumor cell layer at different effect-to-target ratios in each well in RPMI 1640 medium containing 10% FBS supplemented with hIL-2 to reach a final concentration of 100 IU/mL. Luciferase activity was measured after 24 h and 48 h.
4
Transwell Migration Assay of MRC5 Cells
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A total of 8 × 104 TDE-treated MRC5 cells were seeded in the upper chamber of a transwell system (Corning Costar Corp., Armonk, NY, USA). DMEM containing 10% FBS was added to the lower chamber. After 24 h, migrated cells were fixed with 4% polymethanol and stained with 1% crystal violet. Images were captured using a light microscope (SP8; Leica). All experiments were performed in triplicate and repeated three times.
5
Transwell Invasion Assay Protocol
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The measuring of cell invasion activity was carried out in a transwell system (Costar, Cambridge, MA, USA). The transwell chamber contains polycarbonate filters with 8-μm pores coated with Matrigel. The cells were seeded on the upper chamber of transwell and fed with serum-free medium. For the lower chamber, 600 μL complete medium was filled. The non-invasive cells were removed with a cotton swabafter 24–48 h incubation, the number of invasive cells were counted following staining with 1% crystal violet, and cells were counted under light microscope.
6
Cell Viability of SMSCs on 3D Scaffolds
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The cell viability and proliferation was assessed using CCK-8 assay at 1, 3, 7 and 14 days through cocultured with 3D scaffolds in Transwell system (Costar 3422). Briefly, SMSCs suspension with density of 1 × 106 cells ml−1 was injected in 24-well culture plates. 3D scaffolds were placed within the upper chambers. Then the upper chambers were inserted in 24-well culture plates and the complete medium was added. SMSCs seeded in a 24-well culture plate without upper chamber served as control group. The culture plates were incubated at 37 °C in a humidified atmosphere of 5% CO2. The culture medium was exchanged every three days. After 1, 3, 7 and 14 days culture, the culture medium was removed and 400 μl medium containing 40 μl CCK-8 reaction solution was added to each well and incubated for 4 h at 37 °C. Then the medium with CCK-8 was transferred to 96-well tissue culture plate and the absorbance was read at 450 nm using a multidetection microplate reader (MK3, Thermo, USA). All experiments were carried out in triplicate. Then SMSCs were cocultured with 3D scaffolds for 14 days in a tissue culture plate and observed under a phase contrast microscope for cell behavior.
7
Transwell Assay for Breast Cancer Cell Migration
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The migratory activity of breast cancer cells was analyzed through the transwell system (6.5 mm diameter, 8 μM pore size, Corning Costar). Briefly, cells were plated on the upper chamber of transwells at the density of 1 × 104 cells in 100 µL of serum-free DMEM, and complete medium was added to the lower chamber. After 24 h of incubation at 37°C, cells were fixed with methanol followed by staining with hematoxylin and eosin, and counted under a stereo microscope (Carl Zeiss™ Stemi 2000-C). Three independent experiments were performed.
8
Transwell Invasion and Migration Assay
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The Transwell invasion assay was performed as described previously [12 (link)]. Briefly, a total of 4 × 104 cells suspended in 100 μl of serum-free medium were added to the upper chambers of the Transwell system (24 wells, 8-mm pore size; Corning Costar, Lowell, MA, USA) coated with 2 mg/ml Matrigel (BD Biosciences). RPMI-1640 containing 20 % FBS and baicalein (20 and 40 μM) was then added to the lower chamber. After 24 h, the non-invaded cells in the upper chamber were gently removed with a cotton swab, and the cells attached to the lower surface were fixed with precooled methanol and stained with 0.1 % crystal violet. Five fields of each chamber were randomly selected, and the cell numbers were counted under a microscope.
For the migration assay, the cells were seeded into upper chambers that were not coated with Matrigel. The following steps in the assay were the same in the invasion assay. After 24 h, the cells on five randomly selected fields in the lower surface were counted, and the cell numbers were then subjected to statistical analysis.
For the migration assay, the cells were seeded into upper chambers that were not coated with Matrigel. The following steps in the assay were the same in the invasion assay. After 24 h, the cells on five randomly selected fields in the lower surface were counted, and the cell numbers were then subjected to statistical analysis.
9
Transwell Assay for Endothelial Cell Migration
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Migration of endothelial cells was tested using the Transwell system (8 μm pore size and 6.5 mm diameter) in 24-well plates (Corning Costar, Lowell, MA, USA). The lower sides of the filters were coated with 10 μL of type I collagen (0.5 mg/mL). Chemicals were added to the lower chamber in the presence of VEGF (20 ng/mL), and HUVECs (5 × 104 cells/well) were seeded into the upper chamber in serum-free media. The chamber was incubated at 37°C for 24 h. Cells were then fixed with methanol, and stained with hematoxylin (Sigma, St Louis, MO, USA) and eosin (Sigma). Cells on the upper filter surface were removed, and migration was determined using a microscope, at 200× magnification, by counting cells that had migrated to the lower filter side. Samples were assayed twice, in triplicate.
10
Transwell Assay for Cell Migration
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Cell migration was assayed using the Transwell system (Corning Costar, Acton, MA, USA) with 6.5 mm-diameter polycarbonate filters (8-µm pore size). Briefly, the lower surface of the filter was coated with 10 µg/mL fibronectin (Sigma-Aldrich Corp.), 10 µg/mL recombinant human TGFBIp (rhTGFBIp) (Sino Biological Inc., Beijing, China), or bovine serum albumin at a concentration of 3% (w/v) as a control for nonspecific binding. SV40-CECs or primary CECs (105) were seeded onto chemotaxis filters in DMEM plus 1% fetal bovine serum. After the 5-hour migration period, non-migrating cells were removed and migrating cells were stained with hematoxylin and eosin (H&E) and quantified using Kodak 1D software (Eastman Kodak, Rochester, NY, USA). Results are representative of three different experiments in duplicate.
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