Invasion assay was performed using 24-well Transwell polycarbonate membranes (8.0-μm pores; Corning Inc., Corning, NY, USA, Cat#353097), coated with 100μl Matrigel for 30 min. at 37° C, washed with 500 μl PBS, and 1 × 106 cells plated in serum-free media 96 hours after siRNA transfection. Cells were loaded into an invasion chamber with Growth Factor Reduced Matrigel (Corning Cat#354230) diluted 1:10 in appropriate medium. After the 24-hour incubation, migrated cells were fixed and stained with 0.5% crystal violet for 20 minutes. The cells that had penetrated through the membrane were quantified under a microscope at ×40 magnification.
Transwell polycarbonate membrane
Manufactured by Corning
Sourced in United States
Corning Transwell polycarbonate membranes are laboratory equipment designed to facilitate the study of cell migration, transport, and barrier functions. These membranes feature a porous structure that allows for the exchange of media, nutrients, and other substances between the upper and lower chambers of a Transwell insert. The membranes are made of polycarbonate, a durable and transparent material, and are available in a range of pore sizes to accommodate different experimental requirements.
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Lab products found in correlation
Millicell ers, by Merck Group (2 mentions)
Matrigel, by Corning (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Prolong gold mounting media with dapi, by Thermo Fisher Scientific (1 mentions)
Cary eclipse, by Agilent Technologies (1 mentions)
Millicell ers 2 voltohmmeter, by Merck Group (1 mentions)
Matrigel matrix, by Corning (1 mentions)
Three step stain set, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Matrigel, by Merck Group (1 mentions)
Epithelial voltohmmeter, by World Precision Instruments (1 mentions)
Endohm chamber, by World Precision Instruments (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Akt 9272, by Cell Signaling Technology (1 mentions)
Ly294002 s1105, by Selleck Chemicals (1 mentions)
Phospho akt ser473 4060, by Cell Signaling Technology (1 mentions)
Mk2206 s1078, by Selleck Chemicals (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Beyotime (1 mentions)
Cytofix buffer, by BD (1 mentions)
Axiovert 200m fluorescence microscope, by Zeiss (1 mentions)
31 protocols using transwell polycarbonate membrane
1
Transwell Invasion Assay Protocol
2
Imaging PC1 Expression in MDCK Cells
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MDCK cells were grown on Transwell polycarbonate membranes (Corning) for 8–10 days with growth media containing serum below the filter and growth media lacking serum above the filter. Expression of PC1 was induced 16-hours prior to fixation with 50ng/L of doxycycline. Cells were fixed in -20°C methanol for 15 minutes. The methanol was rinsed 2X 2 minutes in TBS. The cells were incubated in cell block and permeabilization buffer (CBP; 2% BSA, 0.2% Triton-X100, 0.05% sodium azide in TBS) for 1 hour at 37°C. Transwells were then cut out and incubated with primary antibodies diluted in CBP overnight at 4°C. The next day they were washed 3X 5 minutes in wash buffer (0.7% fish skin gelatin, 0.05% triton X-100, in TBS) then incubated with fluorescent-conjugated secondary antibodies for 1 hour at 37°C. The Transwells were again washed 3X 5 minutes in wash buffer, followed by two rinses with TBS. The antibodies were fixed to their antigens by post-fixation for 10 minutes in 10% neutral-buffered formalin. They were then washed 2X 5 minutes in TBS and mounted onto a slide and coverslipped using Prolong Gold mounting media with DAPI (Molecular Probes). Images were taken with a Fluoview 1000 confocal laser scanning microscope. Image stacks were processed and analyzed using Image J.
3
Bovine Bronchial Epithelial Cell Culture
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Fresh lungs were collected from calves at a local slaughterhouse. Bovine primary bronchial epithelial cells (PBEC) were isolated as previously described [44 (link)] and were expanded in growth medium (BEGM). When the PBEC had reached confluence, 5 × 105 cells were seeded on the apical compartment of Corning Transwell® polycarbonate membranes with 300 µl medium in the apical compartment and incubated for 24 h at 37°C in a humidified 5% CO2 atmosphere, while the basolateral compartment was filled with 600 µl medium. The transepithelial electrical resistance (TEER) was measured every day by using the Millicell® ERS-2 Voltohmmeter (Millipore) according to the manufacturer’s instructions. Only monolayers with TEER of more than 500 Ω were used for the infection experiments [45 (link)]. Moreover, FITC-labeled 70,000-molecular-weight (Da) dextran (Invitrogen) was added to the apical compartment to measure the integrity of the epithelial barrier. The medium was harvested from the basolateral compartment at different time points, and the fluorescence was determined with a spectrophotometer (Varian Cary Eclipse).
4
Cell Migration and Invasion Assay
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Cell migration and invasion were performed using Corning transwell polycarbonate membranes in 24-well plates (8um). 200 μl of serum-free DMEM cell suspension was inoculated in the upper chamber; 600 μl of DMEM containing 10% FBS was inoculated in the lower chamber; invasion experiments required the addition of a layer of basement membrane extract in the upper chamber. Each chamber uses 50 μl basement membrane extract, which was formed by incubating the matrix gel (Corning Matrigel Matrix) and serum-free DMEM at 37°C in a ratio of 1 to 7 for 30 minutes. After 24h or 48h of cell migration and invasion, cells that migrated into the lower chamber of the polycarbonate filter were fixed and stained with crystal violet. Cells were then observed under a microscope and photographed and counted.
5
Differentiated Bovine Bronchial Epithelial Cells
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Fresh lungs were collected from calves slaughtered at a local slaughterhouse in Germany. Bovine primary bronchial epithelial cells (PBEC) were isolated as previously described [30 (link)] and were expanded in growth medium (BEGM). When the PBEC reached confluence, the cells were transferred to Transwell® polycarbonate membranes (Corning) and maintained under the air–liquid interface (ALI) conditions for at least 4 weeks at 37 °C in a humidified 5% CO2 atmosphere. During this time, the cells grew to a pseudostratified monolayer; as the nuclei were located at different heights, the cells appeared to have a multilayered organization. While all differentiated cells have contact to the filter substrate, there are several basal cells interspersed at the bottom of the monolayer [32 (link)].
6
Mast Cell Migration Assay
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Migration assays were performed using transwell polycarbonate membranes from Costar (8 μm pores, Corning Incorporated, Kennebunk, ME) suitable for mast cells as reported in Kataoka et al. (24 (link)). Briefly, LAD2 cells were starved O/N in complete media without SCF and, when necessary, with biotinylated IgE. The next day, 1 × 105 cells per point were washed with fresh StemPro-34 media and dropped into the upper transwells. After 10 min for stabilization we added the stimulus on the bottom well (100 ng/mL of SCF, 0.4 μg/ml streptavidin or StemPro Supplement 1x), and the cells were left to migrate for 4 h at 37°C and 5% CO2. After 4 h the cells were rinsed with an EDTA-NaCl-PBS buffer to detach them from the bottom of the upper chamber, they were stained with crystal violet and counted under optic microscopy. In all cases, the cells in the upper chamber were counted after 4 h with Trypan blue to evaluate viability.
7
Endothelial-to-Mesenchymal Transition Assay
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Mitral VECs were treated with sham and MI plasma for 24 hours to induce EndMT. The cells were trypsinized, and 20 000 cells in EBM‐2 supplemented with 2% FBS were placed in the upper chamber of 6.5 mm Transwell polycarbonate membranes with 8.0 µm pores (Corning; No. 3422). The lower chambers contained EBM‐2 media with 10% FBS to act as chemoattractant. Cells were allowed to migrate for 12 hours at 37 °C. Cells that migrated through the pores were fixed with methanol and stained with Eosin‐Y, Azure A, and Methylene Blue for visualization and quantification using Three Step Stain Set (Thermo Scientific; No. 22‐050‐272). Cells from 6 different fields in each well were counted in each assay.
8
Cell Migration Assay Protocols with Inhibitors
9
Cell Invasion Assay using Matrigel
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Cell invasion assay was performed as described previously [17 (link)]. Briefly, the membranes of the modified chambers containing Transwell polycarbonate membranes (Costar, Cambridge, MA, USA) were coated with 100 µg/mL Matrigel (Sigma), 1 × 105 cells were seeded into the upper chamber. After 48 h incubation, cells that invaded the Matrigel and polycarbonate membrane to the lower surface were stained with 0.2% crystal violet. The data are presented as the average number of cells attached to the bottom surface from five randomly chosen fields.
10
NMDAR Regulation of Collecting Duct Cells
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mpkCCD cells, an immortal cell line derived from cortical collecting duct transgenic mice, were maintained in culture as described before.(9 (link)) Cells were grown on DMEM/Hams F12 with 2% fetal bovine serum and standard cell culture hormones (Dexamethasone 1 1 mM; mM, T3 1 μM, ITS 100X Gibco cat # 41400045) on 6-well, 4-μm-pore-size Transwell polycarbonate membranes (Costar, Cambridge, MA). All cells were maintained in medium at 37°C, 5% CO2. After monolayers exhibited appropriate transepithelial resistance, the media were aspirated and the cells were washed twice with phosphate-buffered saline. Fully defined media, without FBS or hormones, were then applied to both sides of the monolayer. After 48 hours of incubation in charcoal-stripped media, the experiment was begun. Cells were then treated with vehicle (media), N-methyl-D-aspartate (100 μM, NMDAR agonist) or MK-801 (0.1 mM, NMDAR antagonist). The NMDAR 2C/D selective inhibitor 997-74 (1 μM) was also evaluated.(3 ) Cell monolayer transepithelial voltage (VTE) and resistance (RTE) were measured using an epithelial volt-ohmmeter equipped with stick electrodes (World Precision Instruments, Sarasota, FL). The equivalent transepithelial current (ITE) was calculated according to Ohm's law (ITE = VTE/RTE) and then corrected for the Transwell insert surface area.
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