For cell culture experiments, 1 × 105 tumor cells (Ishikawa + or RL95-2) per well were transferred to a 24-well plate. 1 × 106 PBMC or 1 × 104 Treg were added either in a 0.4-μm-pore Costar Transwell insert (Corning Incorporated, Kennebunk, ME, USA) or directly to the tumor cells without a physical separation. Co-culture was performed at 37 °C for 72 h. PBMC or tumor cells were harvested separately by taking advantage of their different adherence behaviour and washed with PBS for further experiments. Ishikawa + , RL95-2 or PBMC without previous co-culture were chosen as control in each experiment.
Costar transwell insert
Manufactured by Corning
Sourced in United States
Costar Transwell inserts are a type of lab equipment used for cell culture applications. They consist of a permeable membrane insert that fits into a well of a cell culture plate, allowing for the study of cell migration, invasion, and other cell-cell or cell-matrix interactions.
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Lab products found in correlation
Monomac 1 cells, by Leibniz Institute DSMZ (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Matrigel, by BD (3 mentions)
Matrigel invasion chamber, by BD (2 mentions)
Flexstation 3, by Molecular Devices (2 mentions)
L ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Matrigel, by Merck Group (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Cyquant nf cell proliferation assay kit, by Thermo Fisher Scientific (2 mentions)
Diff quick kit, by Siemens (2 mentions)
Cho k1, by Thermo Fisher Scientific (2 mentions)
Mrc 5, by Thermo Fisher Scientific (2 mentions)
F 12k medium, by Thermo Fisher Scientific (2 mentions)
Hek293ft, by Thermo Fisher Scientific (2 mentions)
Cho pgsa 745, by Thermo Fisher Scientific (2 mentions)
Cho pgsb 618, by Thermo Fisher Scientific (2 mentions)
Cho pgse 606, by Thermo Fisher Scientific (2 mentions)
Ep04mm, by Corning (2 mentions)
Bhk 21, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
38 protocols using costar transwell insert
1
Tumor Cell-Immune Cell Co-culture Assay
2
ADSC Migration Assay with Transwell
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The Costar® Transwell®Insert (8 µm, Corning 3422) was employed in migration assay as previously described.30 ADSCs were seeded in the upper chamber in serum‐free medium. The complete medium containing IL‐11 or not were added in the bottom wells. 0.1% crystal violet was used to stain cells in the upper chamber. Cells in the bottom well were counted using inverted optical microscope (ZEISS Group).
3
Cell Migration and Invasion Assays
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Costar Transwell inserts (Corning, USA) and Matrigel Invasion Chambers (BD, USA) were purchased to evaluate the cell migration and invasion respectively [27 (link)]. For migration assay of SW620 cells, transfected cells were harvested and seeded (1× 104 cells per well) onto transwell membranes in serum-free L-15 Medium. Lower chambers of companion plates were filled with 10% FBS L-15 Medium as a chemoattractant. After 24 hours incubation at 37°C, non-migrated cells on the inner surfaces were scrapped with PBS-soaked cotton, while migrated cells were fixed, washed and stained. Five fields were imaged from each transwell membrane using the Live Cell Imaging System (Olympus, Japan), and the number of migrated cells was manually counted. For migration assay of LoVo cells, except for 2 × 105 cells per well and 20 hours of incubation, the rest of the steps are the same as the migration assay of SW620 cells. As for the invasion assays, the test steps are basically the same as above, except for 36 hours of incubation for SW620 cells and 48 hours of incubation for LoVo cells respectively.
4
Measuring Urea Flux in UT-A1 and UT-B Expressing Cells
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MDCK cells stably expressing human UT-A1 or UT-B were cultured in DMEM supplemented with 10% fetal bovine serum. To measure urea flux, cells were grown on 12-mm collagen-coated Costar Transwell inserts (0.4-μm pore size, Corning) for 4 days at 37 °C in the presence of 5% CO2 when cells formed a tight monolayer (transepithelial resistance of 1 kΩ/cm2). An assay of UT-facilitated urea flux was performed as previously described [28 (link)]. After removing the DMEM and washing with PBS, PBS containing forskolin (FSK) with or without CB-20 was added to both the apical- and basal-facing surfaces. After 30 min of incubation, the basal-facing solution was replaced by PBS containing 15 mM urea with or without CB-20. Apical fluid samples were collected at 0, 1, 3, 5, 10, 15, 20, 30, 40, 50, and 60 min. The samples were subjected to an assay for urea (Quantichrome Urea Assay Kit; BioAssay Systems, Hayward, CA) according to the kit procedure. Then, the inhibition rate was calculated from the initial slope with GraphPad Prism 5. The experiment was repeated three times. The UT-B-facilitated urea flux assay was performed with the same procedure as the UT-A1-facilitated urea flux assay, except for FSK stimulation.
5
Transwell Migration and Invasion Assay
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HCT 116 and SW620 cell migration was evaluated by Costar Transwell inserts (Corning, USA) and invasion by Matrigel Invasion Chambers (BD, USA) [25 (link),26 (link)]. For migration assay, transfected cells were serum-starved and then seeded (1× 104 cells per well) onto transwell membranes with serum-free RPMI-1640 medium or L-15 Medium. Lower chambers of companion plates were enriched with 10% FBS medium as a chemoattractant. Incubated for 24 h at 37°C, non-migrated cells on inner surfaces were scrapped, whereas migrated cells were fixed, washed and stained. Five fields were imaged using the Microscopes and Imaging Systems (Leica Microsystems, USA) from each transwell membrane, and the number of migrated cells was counted. As for invasion assays, Matrigel Invasion Chambers was used and performed as migration assay, except for the incubation time was 36 h.
6
Measuring Endothelial Permeability Changes
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To measure permeability changes of endothelial cells in vitro, HUVECs were seeded onto vitronectin (10 g/ml)-coated Costar Transwell inserts (12-mm diameter and 3-μm pore size; Corning) at a cell density of 2 x 104 cells and grown in DEMED with 10% of fetal bovine serum for 2 days to form mature monolayers. Confluent endothelial monolayers were infected in quadruplicate either with SFTSV (MOI = 10), treated with cytokines IL-1β (10 ng/ml) and TNF-α (10 ng/ml), or left untreated (mock). After 48 h postinfection, culture media were replaced and washed with colorless PBS. Then, 500μl Evans blue dye (0.5mg/ml) was added to the upper chamber of monolayers and incubated for 5 min. Liquid (100 μl) from the lower chamber were transferred to a 96-well plate and were read using a spectrophotometric microplate reader at both 620 and 740 nm.
7
Breast Cancer Cell Invasion and Migration Assay
8
Transwell Permeability Assay for HMVEC-L Cells
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HMVEC‐L cells were seeded onto Costar transwell inserts (0.4‐μm pore size; Corning). The following day, rhodamine B isothiocyanate‐dextran (400 μg/mL) was added to the upper wells. After 2 hours of additional incubation at 37°C, the medium in the lower wells was collected, and the fluorescence intensity was measured with 485 and 535 nm as the excitation and emission wavelengths, respectively, using a FlexStation 3 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
9
Influenza Virus Culture and Titration
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Human lung epithelial A549 cells (ATCC CCL-185) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fœtal calf serum and supplemented with 2 mM L-glutamine (Sigma Aldrich), penicillin (100 U/mL), and streptomycin (100 μg/mL) (Lonza), maintained at 37°C and 5% CO2. MucilAir® human airway epithelia (HAE) were obtained from Epithelix SARL (Geneva, Switzerland) and maintained in air-liquid interphase with specific MucilAir® Culture Medium in Costar Transwell inserts (Corning, NY, USA) according to the manufacturer's instructions.
Influenza viruses A/Lyon/969/09 and A/Quebec/144147/09 were produced in MDCK (ATCC CCL-34) cells in EMEM supplemented with 2 mM L-glutamine (Sigma Aldrich), penicillin (100 U/mL), streptomycin (100 μg/mL) (Lonza) and 1 μg/mL trypsin. Viral titers in plaque forming units (PFU/ml) and tissue culture infectious dose 50% (TCID50/mL) were determined in MDCK cells as previously described (29 (link), 30 (link)).
Influenza viruses A/Lyon/969/09 and A/Quebec/144147/09 were produced in MDCK (ATCC CCL-34) cells in EMEM supplemented with 2 mM L-glutamine (Sigma Aldrich), penicillin (100 U/mL), streptomycin (100 μg/mL) (Lonza) and 1 μg/mL trypsin. Viral titers in plaque forming units (PFU/ml) and tissue culture infectious dose 50% (TCID50/mL) were determined in MDCK cells as previously described (29 (link), 30 (link)).
10
In vitro Migration Assay for Cells
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In vitro migration assay was performed using Costar Transwell inserts (Pore size: 8 μm; Corning, Albany, NY) in 24-well plates as described previously [48] (link)–[50] (link). Approximately 1 × 104 cells in 200 μl of serum-free medium were placed in the upper chamber, and 300 μl of the same medium containing ATP was placed in the lower chamber. Before performing the migration assay, cells were pre-treated for 60 min with nicardipine followed by treatment with ATP during the 24-h migration assay (incubated at 37°C in 5% CO2). After the 24-h assay, the cells were stained with 0.05% crystal violet and 2% methanol. Non-migratory cells on the upper surface of the filters were removed by wiping with a cotton swab. Cell number was counted in five random fields per well under a microscope at 200× magnification. Images of migratory cells were observed and acquired using a digital camera and light microscope.
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In vitro migration assay was performed using Costar Transwell inserts (Pore size: 8 μm; Corning, Albany, NY) in 24-well plates as described previously [48] (link)–[50] (link). Approximately 1 × 104 cells in 200 μl of serum-free medium were placed in the upper chamber, and 300 μl of the same medium containing ATP was placed in the lower chamber. Before performing the migration assay, cells were pre-treated for 60 min with nicardipine followed by treatment with ATP during the 24-h migration assay (incubated at 37°C in 5% CO2). After the 24-h assay, the cells were stained with 0.05% crystal violet and 2% methanol. Non-migratory cells on the upper surface of the filters were removed by wiping with a cotton swab. Cell number was counted in five random fields per well under a microscope at 200× magnification. Images of migratory cells were observed and acquired using a digital camera and light microscope.
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