To evaluate the putative effects of serum from acutely YFV-infected patients on endothelial barrier function, we used the TEER assay as described previously (26 (link), 27 (link)). In brief, human umbilical vein endothelial cells, kindly donated by Dr. Miriam Fonseca-Alaniz (Instituto do Coração, InCor, University of São Paulo, Brazil) were seeded (6×104 cells/well) in Transwell polycarbonate membrane inserts (0.4 μm pore, 6.5 mm diameter; Corning Inc.) in endothelial cell growth basal medium 2 supplemented with an Endothelial Cell Growth Medium-2 (EGM-2TM) supplemental bullet kit (Lonza). After 72h of incubation at 37°C and 5% CO2, cells were treated with human sera (10% final vol/vol concentration) obtained from YFV-positive severe and non-severe patients or YFV-negative blood donors (healthy controls). TEER values, expressed in Ohms (Ω), were collected at sequential 2-h time-points 2–10h following treatments using an Epithelial Volt Ohm Meter (EVOM) with a “chopstick” electrode (World Precision Instruments). Resistance of inserts with no cells (blank) and inserts with cells (untreated) containing medium alone, were used to calculate relative TEER as a ratio of the corrected resistance values as (Ω experimental condition - Ω blank)/(Ω untreated - Ω blank). Recombinant YFV NS1 (Native Antigen Co.) at 10 μg/mL was used as positive control.
Transwell polycarbonate membrane insert
Manufactured by Corning
Sourced in United States
Transwell polycarbonate membrane inserts are a type of lab equipment used for cell culture applications. The inserts feature a polycarbonate membrane that allows for the study of cell migration, permeability, and co-culture experiments. The membrane provides a barrier between the upper and lower chambers, enabling researchers to observe and analyze cellular interactions and processes.
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Lab products found in correlation
Matrigel, by BD (2 mentions)
Calcein, by Thermo Fisher Scientific (2 mentions)
Chopstick electrode, by World Precision Instruments (1 mentions)
Epithelial voltohmmeter, by World Precision Instruments (1 mentions)
Endothelial cell growth medium 2 egm 2tm supplemental bullet kit, by Lonza (1 mentions)
Growth factor reduced matrigel, by BD (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions)
Celltracker cm dii dye, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Evos microscope, by Thermo Fisher Scientific (1 mentions)
White 96 well plate, by Corning (1 mentions)
Gibco hank s balanced salt solution hbss, by Thermo Fisher Scientific (1 mentions)
Ultima gold, by PerkinElmer (1 mentions)
Cremophor rh40, by BASF (1 mentions)
3h digoxin, by PerkinElmer (1 mentions)
Culture insert, by Ibidi (1 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Synergy mx, by Agilent Technologies (1 mentions)
Crystal violet, by Beyotime (1 mentions)
25 protocols using transwell polycarbonate membrane insert
1
Evaluating Endothelial Barrier Function in Yellow Fever
2
Transwell Migration and Invasion Assay
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Cell migration and invasion were assessed in Transwell polycarbonate membrane inserts (Corning) as previously described [59 (link)]. For the invasion assay, the transwell membranes were coated with Growth Factor Reduced Matrigel (12.5 μg in 60 μl /well (BD Biosciences). After overnight incubation at 37° C, cells that had migrated or invaded the underside of the membrane were stained with crystal violet. The total membrane was scanned and analyzed using ImageJ software.
3
Co-culture Models for Cancer Angiogenesis
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Endothelial cell/cancer cell (EC) co-cultures, pericyte/cancer cell (PC) co-cultures, and pericyte/endothelial cell/cancer cell (PEC) co-cultures were established using EA.hy926, LLC, and C3H/10T1/2 cells. For the EC co-cultures, LLC cells (2.5 × 105 cells/well) were first seeded in 12-well plates and allowed to adhere for 4 h, after which 12-well Transwell polycarbonate membrane inserts (3 μm; Corning, NY, USA) were placed above each well. EA.hy926 cells (5 × 105 cells/well) were seeded over the Transwell membrane inserts and incubated at 37 °C for 48 h. For PC co-cultures, LLC (2.5 × 105 cells/ well) cells were seeded onto the 12-well plate surface, while C3H/10T1/2 cells (2.5 × 105 cells/well) were seeded above the Transwell membrane inserts. For the PEC co-cultures, LLC (2.5 × 105 cells/ well) cells were seeded onto the 12-well plate surface, and C3H/10T1/2 cells (2.5 × 105 cells/well) were added to the lower surface of the Transwell membrane inserts and was followed by the addition of EA.hy926 cells (5 × 105 cells per well) onto the upper surface of the Transwell membrane inserts. The other procedures remained the same.
4
HUVEC Transwell Migration Assay
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A total of 1x105 HUVECs were seeded on Transwell polycarbonate membrane inserts (8.0-µm pores; Corning, Inc.) and maintained in 100 µl high-glucose DMEM without serum. NP or DNP cells were cultured in complete medium with melatonin (10 µM) or TGF-β1 (10 ng/ml) in the lower chamber. The groups were as follows (upper chamber/lower chamber): HUVECs/NP, HUVECs/DNP, HUVECs/DNP + M, HUVECs/NP + V, HUVECs/NP + M + V, HUVECs/NP + V + T. All inserts were incubated for 24 h at 37˚C and 5% CO2 in an incubator. The inserts were washed with PBS and the cells on the top surface of the inserts were carefully removed using a cotton swab. The inserts were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by staining with 0.1% crystal violet for 30 min at room temperature. Migrated cells were observed and counted under x40 magnification with 5 fields randomly selected in each sample using a light microscope (Olympus Corp.).
5
Quantifying Cell Invasion Potential
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Cell invasion was studied using Corning Transwell polycarbonate membrane inserts with 24 pores (pore size 8.0 μm, membrane diameter 6.5 mm). The inserts were set into 24-well plates to form two champers, and the upper chambers were precoated with Matrigel Basement Membrane Matrix (BD Biosciences-Discovery Labware). HCCLM3 cells in the logarithmic growth phase were trypsinized and resuspended in nonserum containing DMEM media. Cells in 300 μl DMEM media with a density of 5 × 103 cells/ml were plated in the upper chambers. 500 μl DMEM medium with 10 % FBS was used as a chemoattractant in the lower chamber. Cell invasion was allowed to progress for 24 h at 37 °C with 5 % CO2. Afterward, the Matrix gel and cells on the top membrane surface were removed with a cotton swab. Transwell membranes were then stained with crystal violet, and the light microscopy was used to photograph the cells that migrated through the membrane to the lower surface. The migrating cells were quantified by dissolving the purple crystals on the membranes in 500 μl 10 % acetic acid, and measuring their OD values at 570 nm by Multiskan Ascent.
6
Optimizing iBMEC Culture Conditions
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On day 8, the collagen IV/fibronectin-coated transparent polyester ThinCert® inserts (12-well format, 1.131 cm2, 0.4 µm pore size, 2 × 106 pores/cm2, i.e., approx. 0.25%, porosity, Greiner Bio-One GmbH #665641; purchased from In Vitro A/S, Fredensborg, Denmark) were seeded with iBMECs at four different seeding densities (250,000; 500,000; 750,000, and 1,000,000 cells/cm2). On day 9, the medium was changed to hECSR2 (hECSR1 without RA or hFGF) and the cells were used for the experiment on day 10 or 11 in case the TEER was not sufficiently high on day 10. It should be noted that during the differentiation protocol optimisation, Corning® Transwell® polycarbonate membrane inserts (12-well format, 1.12 cm2, 0.4 µm pore size, 1 × 108 pores/cm2, i.e., approx. 12.57%, porosity, Corning #3401) were used.
7
BeWo b30 Transwell Culturing Protocol
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BeWo b30 cells were cultured on Transwell® polycarbonate membrane inserts (24-well format; 0.4 μm pore size, 0.33 cm2 cell growth area, 200 μL apical volume, 1′000 μL basolateral volume; Corning, Sigma-Aldrich) at a density of 60′000 cells/well. These inserts were then cultivated in a cellZscope (nanoAnalytics) at 37°C/5% CO2. Cell culture medium was replaced every 2 days up to 11 days to find the best possible conditions.
8
Transmigration Assay for Endothelial Cells
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Transmigration assay was performed as previously described (Seehaus et al., 2009 (link)). In brief, Corning® Transwell® polycarbonate membrane inserts (6.5 mm diameter, 8 μm pore size) were used. Endothelial monolayer was incubated with Ang II 24 h after transfected with FKBP11-Si RNA or NC-Si RNA for 24 h. Then 5 × 105 THP-1 cells were seeded into each upper compartment containing 100 μL serum-free RPMI medium. In preincubation experiments cells were washed twice before THP-1 cells were added to the endothelial cell monolayer. Following incubation at 37°C in a humidified 5% CO2 incubator for 12 h the upper compartments were removed and THP-1 cells transmigrated into the lower compartment were counted after stained with crystal violet. Each assay was performed in triplicates and repeated at least three times. Cells were counted in five randomly selected squares per well and presented as the number of migrated cells/field.
9
Adipocyte Progenitor Migration Assay
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Migration assays were performed using Transwell polycarbonate membrane inserts with a 5‐μm pore diameter purchased from Corning (Corning, NY). THP‐1 cells were stained with CellTracker CM‐DiI dye (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's directions and then plated into the inserts. Nonsenescent control or senescent human primary adipocyte progenitors were seeded into the bottom chambers of the plates. After 24 hr, DiI‐labeled THP‐1 cells that had migrated into the bottom chamber were imaged.
For neutralizing antibody experiments, THP‐1 cells (105) were plated into Transwell inserts and allowed to migrate into the bottom chamber for 6 hr at 37°C. After 6 hr, the number of cells that had migrated into the lower chamber was quantified using the CyQUANT Cell Proliferation Assay Kit (Life Technologies) according to the manufacturer's instructions. All neutralizing antibodies were purchased from R&D. The concentrations for the antibodies in the CM are Goat IGG (1 mg/ml), M‐CSF (1 mg/ml), MCP1 (0.2 mg/ml), and MIP‐1b (0.2 mg/ml).
For neutralizing antibody experiments, THP‐1 cells (105) were plated into Transwell inserts and allowed to migrate into the bottom chamber for 6 hr at 37°C. After 6 hr, the number of cells that had migrated into the lower chamber was quantified using the CyQUANT Cell Proliferation Assay Kit (Life Technologies) according to the manufacturer's instructions. All neutralizing antibodies were purchased from R&D. The concentrations for the antibodies in the CM are Goat IGG (1 mg/ml), M‐CSF (1 mg/ml), MCP1 (0.2 mg/ml), and MIP‐1b (0.2 mg/ml).
10
Transwell Cell Migration Assay
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A total of 10,000 cells in 100 μl serum-free DMEM containing DMSO, PTX, GPT, or combination were added into Corning Transwell polycarbonate membrane inserts coated with Matrigel (300 μg/mL). And medium containing 10% FBS was added to the bottom chamber. After 24 h incubation, the cells that remained on the above surface of the insert membrane were scraped off with a cotton swab. The cells that passed through Matrigel to the bottom of the insert were fixed with paraformaldehyde and stained with 0.1% crystal violet in methanol. The inserts were photographed, and the cells were counted.
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