For the cell invasion assay, 1 × 104 cells in 100 μL serum‐free medium were seeded in a transwell apparatus (Costar, Corning, NY, USA) containing a fibronectin‐coated polycarbonate membrane insert. Medium (500 μL) containing 10% fetal bovine serum was added in the lower chamber as chemoattractant. After the 8‐h incubation at 37°C in a 5% CO2 atmosphere, the insert was washed extensively with PBS and the top surface of the insert were wiped clean with a cotton swab to remove cells. Cells on the lower surface were fixed with methanol, stained with crystal violet. Cells adhering to the lower surface were counted using five predetermined fields (×100). All assays were independently repeated in triplicates.
Transwell apparatus
Manufactured by Corning
Sourced in United States, China
The Transwell apparatus is a cell culture system that allows for the separation of different cell types or cellular compartments within a single culture. It consists of an upper chamber and a lower chamber, separated by a porous membrane. This system enables the study of cell-cell interactions, transport processes, and barrier functions in a controlled in vitro environment.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (34 mentions)
Matrigel, by R&D Systems (20 mentions)
Matrigel, by Corning (7 mentions)
Boyden chamber, by Corning (3 mentions)
Matrigel coated polycarbonate membrane insert, by Corning (2 mentions)
Transwell chamber, by BD (2 mentions)
Dmil1, by Leica (2 mentions)
Crystal violet, by Merck Group (2 mentions)
Crystal violet, by Beyotime (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Matrigel, by Merck Group (2 mentions)
Edu cell proliferation assay kit, by RiboBio (2 mentions)
Eclpse 80i system, by Nikon (2 mentions)
Giemsa, by Merck Group (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
Nis elements d 4, by Nikon (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
Hdmec, by ScienCell (1 mentions)
Dve00, by R&D Systems (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
158 protocols using transwell apparatus
1
Cell Invasion Assay Protocol
2
Transwell Assays for Cell Migration and Invasion
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For cell migration assays, we added 500 μl DMEM containing 10% FBS to each well of a 24-well plate, resuspended 5 × 104 cells in 200 μl DMEM without FBS, and finally seeded these cells into the upper chamber of the Transwell apparatus (Costar, Corning Inc., Corning, NY, USA). For the invasion assay, 500 μl of DMEM containing 10% FBS was added to the 24-well plates per well. Then we applied Matrigel matrix to the upper chamber of the Transwell apparatus, resuspend 1 × 105 cells in 200 μl DMEM without FBS and seeded them into the upper chamber. After 24 hours, we fixed these Transwell inserts with methanol at room temperature for 15 minutes, stained them with 0.1% crystal violet at room temperature for 15 minutes, washed them with PBS three times and observed them under a microscope.
3
Osteogenic Differentiation of PDLSCs under IL-1β
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PDLSCs were plated at 3 × 104 cells per well in the lower transwell chamber and cultured to 80% confluence in culture medium (α-MEM with 10% FBS and 1% antibiotic). The medium was replaced with an osteogenic medium containing 0, 0.01, 0.05, 0.25, 1.25 or 6.25 ng/ml IL-1β and cells were cultured for 24 h. An osteogenic medium with 0, 0.01, 0.05, 0.25, 1.25 or 6.25 ng/ml IL-1β and without PDLSCs used as a control. A total of 3 × 104 RAW 264.7 cells were plated on a Matrigel-coated polycarbonate membrane insert (8.0 mm pores) in a transwell apparatus (Costar, Shanghai, China) and maintained in 100 μl of complete medium (α-MEM with 10% FBS and 1% antibiotic). Then, the inserts were washed with PBS, and the cells on the top surface of the insert were removed by wiping the surfaces with a cotton swab. The cells that migrated to the bottom surface of the insert were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and then subjected to a microscopic inspection and cell count. The cells were counted under × 200 magnification. Every sample was counted in five randomly chosen fields and the values were averaged.
4
Transwell Migration Assay Protocol
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Cells were seeded, in pure RPMI1640 (1×104 cells/0.25mL/well), onto the upper well, and a 6.5-mm pore-size polycarbonate membrane chamber was inserted into the transwell apparatus (Costar, MA, USA). RPMI1640 containing 10% FBS was added into the lower well. Cells were incubated and migrated to the bottom surface after 24 hours, fixed, stained, rinsed and examined by inverted microscopy.
5
Endothelial Migration Assay using PDAC Conditioned Media
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For endothelial migration assays, conditioned media (CM) were harvested from PDAC cells cultured in 60-mm plates in RPMI-1640 without FBS for 24 hours. The media were added to the bottom chambers of 24-well tissue culture plates in triplicate. HUVEC (40,000 cells) were seeded into Boyden chambers (8 μm pore size with polycarbonate membrane). The chambers were then inserted into a transwell apparatus (Costar, Cambridge, MA, USA). Cells were allowed to migrate for 12 h, and then fixed and stained in 0.3% crystal violet for 30 mins. After rinsing with PBS, the migrated cells were subjected to microscopic inspection.
6
Cell Migration and Invasion Assay
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A scratch was made on 80% confluent monolayer cell cultures, and culture medium was replaced with serum-free DMEM. The healing process was monitored for 24 h, and the percentage of wound closure was calculated using ImageJ software (version 1.51; National Institutes of Health).
In total, 5×104 cells in 200 µl serum-free DMEM medium were seeded onto the upper chamber of Transwell apparatus (Costar; Corning, Inc.) with a Matrigel-coated membrane. A total of 600 µl DMEM containing 10% FBS was added to the lower chamber as a chemoattractant. After a 12-h incubation at 37°C in a 5% CO2. atmosphere, the cells that invaded the lower chamber through the membrane were fixed with 4% methanol for 30 min at room temperature, stained with 0.1% crystal violet for 15 min at room temperature and counted under a light microscope (magnification, ×100).
In total, 5×104 cells in 200 µl serum-free DMEM medium were seeded onto the upper chamber of Transwell apparatus (Costar; Corning, Inc.) with a Matrigel-coated membrane. A total of 600 µl DMEM containing 10% FBS was added to the lower chamber as a chemoattractant. After a 12-h incubation at 37°C in a 5% CO2. atmosphere, the cells that invaded the lower chamber through the membrane were fixed with 4% methanol for 30 min at room temperature, stained with 0.1% crystal violet for 15 min at room temperature and counted under a light microscope (magnification, ×100).
7
In Vitro Cell Invasion Assay
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The in vitro cell invasion of MDA231 cells was assessed using the transwell assay according to the manufacturer’s protocol 12 . In brief, 5 × 105 cells incubated with 500 μg of platelet microvesicles were seeded into the upper chamber of the transwell apparatus (Corning Costar, Waltham, MA, USA), which was precoated with 50 μL of a Matrigel solution in serum‐free medium, and medium supplemented with 15% FBS was added to the bottom chamber. After 24 h, the cells on the upper surface that did not pass through the 8‐μm pore‐size polycarbonate filter were removed using a moistened cotton swab; the cells migrating to the lower membrane surface were fixed in 100% methanol for 20 min, stained with 0.4% crystal violet for 20 min and counted under a microscope (Nikon, Tokyo, Japan) at 100× magnification.
8
Transwell Assay for Cell Migration and Invasion
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Cell migration assay was carried out as follows. In a Transwell apparatus (CoStar, USA), 1 × 104 cells were plated on fibronectin-coated polycarbonate membrane inserts in 200 μL DMEM without FBS. In the lower chamber, 800 μl DMEM containing 10% FBS was supplemented. Following an 8 h incubation in a 5% CO2 incubator at 37 °C, the insert underwent PBS washes, and cells on its top surface were removed with a cotton swab. Then, cells attached to the lower surface underwent methanol fixation, Giemsa staining, and counting by microscopy. The cell invasion assay was similarly performed, with the Transwell membrane precoated with Matrigel (R&D Systems, USA) at 24 μg/μl. All trials were repeated independently at least 3 times.
9
Cell Migration and Invasion Assay
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For the cell migration assay, 1 × 104 cells in 100μl medium without fetal bovine serum were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Costar, Corning, NY, USA). In the lower chamber, 500μl medium with 10% fetal bovine serum was added as chemoattractant. After the cells were incubated for 6h at 37°C in a 5% CO2 atmosphere, the insert was washed with phosphate buffered saline, and cells on the top surface of the insert were removed with a cotton swab. Cells adhering to the lower surface were fixed with methanol, stained with crystal violet solution and counted under a microscope in five predetermined fields (×100). All assays were independently repeated at least thrice. The procedure for the cell invasion assay was similar to the cell migration assay, except that the transwell membranes were precoated with 24μg/μl matrigel (R&D Systems, Inc., Minneapolis, MN, USA) and the cells were incubated for 8h at 37°C in a 5% CO2 atmosphere. Cells adhering to the lower surface were counted the same way as the cell migration assay.
10
Cell Migration and Invasion Assays
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For cell migration assays, 1 × 105 cells in 100 µl DMEM medium without FCS were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Costar, MA). In the lower chamber, 600 µl DMEM with 10% FCS was added as chemoattractant. After the cells were incubated for 16 h at 37 ℃ in a 5% CO2 atmosphere, the insert was washed with PBS, and cells on the top surface of the insert were removed with a cotton swab. Cells adhering to the lower surface were fixed with methanol, stained with Giemsa solution, and counted under a microscope in five predetermined fields (×200). All assays were independently repeated three times. For the cell invasion assay, the procedure was similar to the cell migration assay, except that the transwell membranes were precoated with 24 µg/µl Matrigel (R&D Systems, USA).
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