The cells were transfected and treated as described above. Then, 1 × 105 cells/well were seeded in 24-well plates with Transwell polycarbonate filters using 6.5 mm diameter inserts with 8 μm pores (Corning Life Sciences, Amsterdam, The Netherlands). The cells were cultured inside of each insert with 300 μl 1% FCS culture medium and in the lower well of the migration plate with either 500 μl 10% FCS, 1% FCS in the negative control or 20% FCS in the positive control. After 48 h of incubation, the cells were fixed with 4% paraformaldehyde, followed by staining with crystal violet. The nonmigratory cells on the interior of the insert were gently wiped off with a cotton swab. The migratory cells remained on the bottom of the insert membrane. The Transwell inserts were washed twice with PBS to remove unbound crystal violet and then air-dried. The migratory cells on the bottom of the insert membrane were examined microscopically at ×200 magnification. For analysis, crystal violet was eluted using 33% (v/v) acetic acid with dd H2O. The bound crystal violet was eluted by adding 400 μl of 33% acetic acid into each insert and shaking for 10 min, and 100 μl of the eluent was transferred to a 96-well plate and quantified by measuring the absorbance at 590 nm with a plate reader.
Polycarbonate transwell filter
Manufactured by Corning
Sourced in United States
Polycarbonate transwell filters are a laboratory equipment product designed to facilitate cell culture and migration studies. These filters provide a permeable membrane support that allows for the controlled movement of cells, molecules, and other substances between different compartments of a cell culture system.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (11 mentions)
Matrigel, by Corning (5 mentions)
Crystal violet, by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Millicell ers 2 voltohmmeter, by Merck Group (3 mentions)
Millicell ers 2, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Matrigel basement membrane matrix, by BD (2 mentions)
4 kda fitc dextran, by Merck Group (2 mentions)
Evom voltohmmeter, by World Precision Instruments (1 mentions)
Eclipse ts100 microscope, by Nikon (1 mentions)
Bovine plasma fibronectin, by Merck Group (1 mentions)
Calf skin collagen type 1, by Merck Group (1 mentions)
Recombinant human tumor necrosis factor alpha tnf α, by Thermo Fisher Scientific (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
Rabbit anti e cadherin, by Cell Signaling Technology (1 mentions)
Hoechst 33342 stain, by Thermo Fisher Scientific (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Eclipse ti s microscope, by Nikon (1 mentions)
71 protocols using polycarbonate transwell filter
1
Transwell Migration Assay Protocol
2
Endothelial Barrier Permeability Assay
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Endothelial cells were grown to confluence on Transwell polycarbonate filters (Corning Inc., Corning, NY, United States) mounted in a chamber insert. Resistance across cells was monitored daily using an EVOM volt-ohmmeter (World Precision Instruments). The lipids, 2-ClHDA or HDA, (10 μM) were added to each well then the resistance across each well was monitored up to 24 h.
3
Transwell-based Cell Invasion Assay
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Matrix intrusion tests were performed in 24-well plates containing Transwell polycarbonate filters (Corning, New York, NY, USA) with an aperture of 8 μm. To the lower chamber, 600 μl medium containing 10% fetal bovine serum (FBS) was added. The cells were placed in serum-free medium (1 × 104 cells/100 μl) in Matrigel (BD Biosciences, San Jose, CA, USA) coated upper chambers and incubated at 37 °C for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Finally, the invaded cells were photographed and counted.
4
Cell Transmigration Assay Protocol
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Cells (105 cells/cm2) were seeded in 24-well plates with transwell polycarbonate filters (8 mm, Corning Life Sciences, Amsterdam, The Netherlands). The number of transmigrated cells was counted under a Nikon Eclipse TS100 microscope.
5
Transendothelial Migration Assay for PBMCs
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hCMEC/D3 cells were grown (72 h) on Transwell polycarbonate filters (membrane diameter 6.5 mm, 5 μm porosity—Corning, Germany) previously coated with calf skin collagen type I (Sigma-Aldrich) and bovine plasma fibronectin (Sigma-Aldrich). Transmigration assays were run with PBMCs in their autologous sera (AS) or with a swap of sera, i.e., healthy donor PBMC + AS or healthy donor PBMC + RRMS patient sera. EBM-2 complete medium (w/o VEGF) containing 20% sera was added to the lower compartment of Transwell 30 min prior to transmigration assay. Then, PBMCs (1x106 cells) were suspended in EBM2 medium containing sera and added to the upper compartment of Transwell in contact with hCMEC/D3 monolayer. The transmigration assays were run during 4 h at 37 °C at 5% CO2. Adhered cells were detached from the top compartment of Transwell by using a 0.2% trypsin solution, whereas transmigrated cells were collected from the bottom compartment by aspirating the medium. Cells were fixed with 2% PFA (10 min at room temperature), and staining was performed for further FACS analysis on the transmigrated cells.
6
Endothelial Dysfunction in Co-Culture Model
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Firstly, human umbilical vein endothelial cells (HUVECs, the kind gift from the Atherosclerosis Research Centre, Nanjing Medical University, Nanjing, People’s Republic of China) were seeded onto gelatin-coated Transwell polycarbonate filters (0.4 µm pore, 6.5 mm membrane diameter; Corning Incorporated, Corning, NY, USA) and cultured for 3 days to form a restrictive endothelial monolayer. Then, RAW264.7 macrophage cell line (iMΦ) in logarithmic growth phase was plated into the lower compartments of Transwell chambers and co-cultured with HUVECs on the upper compartment of Transwell chambers for a further 24 h. To accelerate endothelial dysfunction, the culture medium was replaced with serum-free M199 containing 10 ng/mL human recombinant tumor necrosis factor-alpha (TNF-α) (PeproTech, Rocky Hill, NJ, USA) and 40 µg/mL oxidized low-density lipoprotein (ox-LDL) (Yiyuan Biotech, Guangzhou, People’s Republic of China).35 (link),36 (link) Finally, the expression of CD44 receptors on HUVEC monolayer and the permeability of HUVEC monolayer after stimulation with TNF-α and ox-LDL for 12 h were analyzed to validate the formation of the injured HUVECs/iMΦ co-culture system.
7
CRC Cell Lines: Culturing and Drug Treatments
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CRC cell lines (Caco-2, HCT-116, and HT-29) were obtained from the American Type Culture Collection (ATCC) and were authenticated using their short tandem repeat (STR) profiles. Cells were cultured at 37°C in a humidified atmosphere of 5% CO2/air in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Inc.), penicillin G (60 mg/l), and streptomycin (100 mg/l). For experimental purposes, the cells were seeded in culture flasks or on plates, glass coverslips, or Transwell polycarbonate filters with a 0.4-µm pore size (Corning, Inc.). After reaching 80% confluence, the cells were treated with the appropriate drugs for 24 h. The drugs used for the different assays were tunicamycin at 0.25, 0.50, 0.75, and 1 µg/ml; PD153035 at 1, 10, and 20 µg/ml; OSI906 at 2, 4, and 8 µg/ml; U73122 at 7, 14, and 21 µg/ml; PD98059 at 7, 14, and 21 µg/ml; LY294002 at 2, 4, and 6 µg/ml; STA-21 at 3, 4, 5, 6, and 7.5 µg/ml; H-89 at 9 µg/ml; and forskolin at 4 µg/ml.
8
Ultrastructural Analysis of Cell Monolayers
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Cells were cultured on Transwell polycarbonate filters (Corning, Inc.) and fixed for 60 min on the apical side of the monolayer with a solution containing 2.5% glutaraldehyde, 1% freshly prepared paraformaldehyde, 8% sucrose, 2 mM CaCl2, and 6 mg/ml ruthenium red in 0.1 M cacodylate buffer, pH 7.4. After washing with cacodylate buffer containing ruthenium red for 10 min, the cells were postfixed with 1% OsO4 and 6 mg/ml ruthenium red in cacodylate buffer for 45 min. The cell monolayers were then washed with cacodylate buffer, dehydrated with an acetone series, and embedded in Epon resin. Ultrathin sections (70 nm) were stained with lead citrate and observed with a Zeiss CEM-900 transmission electron microscope (Carl Zeiss).
9
Immunofluorescence Staining of PC1 Protein
10
Measuring TEER in MDCK II cell cultures
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MDCK II cells were cultured on Transwell polycarbonate filters (0.4-µm pore size; #3413; Corning) for 5–7 d. After cells were equilibrated to RT, the electric resistance between the apical and basolateral chambers was measured using a Millicell ERS-2 electrical resistance system (Merck Millipore). Blank measurements were performed on Transwell filters without cells. After subtraction of the mean blank values, the electric resistance was multiplied by the growth area of the Transwell filter to yield the unit area resistance (Ω · cm2). Graphs were generated using Excel. Statistical tests were performed using Excel.
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