Human umbilical vascular endothelial cells (HUVECs) were seeded on the inner chamber of collagen-coated Transwell filters (0.33-cm2, Corning Life Sciences) in M199 medium (Biowhittaker) supplemented with 2 mM L-glutamine, 10% FBS, 10μg/ml endothelium mitogen (Fisher), 20μg/ml heparin sodium salt (Sigma) and 100 U penicillin/streptomycin following a previously described protocol [68 (link)]. The cell monolayer was allowed to form over 4–5 days.
Collagen coated transwell filters
Manufactured by Corning
Sourced in United States
Collagen-coated Transwell filters are a type of lab equipment used for cell culture applications. The filters provide a porous membrane coated with collagen, which facilitates the growth and differentiation of cells that require a suitable extracellular matrix. The filters can be used to study cell migration, permeability, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by American Type Culture Collection (2 mentions)
M199 medium, by Lonza (1 mentions)
Heparin sodium salt, by Merck Group (1 mentions)
Bronchial epithelial growth medium (begm), by Lonza (1 mentions)
T 75 flasks, by Corning (1 mentions)
Vx 809, by Selleck Chemicals (1 mentions)
Collagen type 4, by Merck Group (1 mentions)
Evom2, by World Precision Instruments (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Prism 9, by GraphPad (1 mentions)
Evom2 voltohmmeter, by World Precision Instruments (1 mentions)
Spectramax plate reader, by Molecular Devices (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
8 protocols using collagen coated transwell filters
1
Cultivating HUVEC Monolayer on Transwell
2
Polarized Primary RPE Culture Generation
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Primary RPE were harvested from porcine eyes as previously described (Toops et al., 2014 (link)), plated onto T25 flasks in DMEM with 10% FBS for the first week and 1% FBS for the second week (American Type Culture Collection, Manassas, VA). To generate polarized cultures, cells were plated at ∼350,000 cells/cm2 onto collagen-coated Transwell filters (Corning, Corning, NY) (Toops et al., 2014 (link)). Primary RPE were magnetofected with 0.5 µg mito-GCaMP5G plasmid (105,009, Addgene, Watertown, MA) for every ∼3 × 105 cells using LipoMag Transfection Kit (OZ Biosciences, San Diego, CA).
3
Cystic Fibrosis CFTR Trafficking Correction
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T84 cells were obtained from the ATCC (Manassas, VA), grown in T75 flasks (Costar, St. Louis, MO), seeded onto collagen-coated Transwell filters (6.5 mm; Corning, Inc., Corning, NY), and maintained in DMEM medium containing 10% FBS (Life Technologies, Grand Island, NY). Resistance was monitored using EVOM2 (World Precision Instruments, Sarasota, FL), and cells were studied when resistance was at 500–1000 Ω.cm2. Cells were serum starved (0.1% FBS) for 24 h prior to TGF-beta exposure. HAECs obtained from the University of North Carolina Airway Cell Core (under the direction of Dr. Scott Randell) were isolated from donor or recipient lungs that were cryopreserved at passage 1 and cultured as previously described [29] (link). Briefly, HAECs were cultured in Bronchial Epithelial Growth Medium (BEGM; Lonza, Hopkinton, MA) on Purecol-coated tissue-culture dishes (Advanced Biomatrix, San Diego, CA) until 80–90% confluent, then passaged and plated onto Transwell-Clear permeable supports (0.4 µm pore size; Corning, Inc.) coated with type IV collagen (Sigma-Aldrich, St. Louis, MO). To correct F508del CFTR trafficking, F508del/F508del HAECs were treated with VX-809 (3 µM; Selleck Chemicals, Houston, TX) for 48 h prior to study [27] (link), [28] (link).
4
Transmigration Assay for Neutrophil Migration
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Transmigration assay was performed as previously described (Bou Ghanem et al., 2014 (link)). Briefly, Human pulmonary mucoepidermoid carcinoma-derived NCI-H292 (H292) (ATCC) cells were seeded on inverted Transwell filters collagen-coated Transwell filters (0.33-cm2, Corning Life Sciences) and then allowed to grow and polarize for 1 week in RPMI 1640 medium (ATCC) with 2 mM L-glutamine, 10% FBS (Invitrogen) and 100 U penicillin/streptomycin. On the day of the migration assay, the epithelial monolayers were washed out of the antibiotic-containing media and equilibrated in HBSS for 30 min. The apical side of monolayers was infected with S. pneumoniae at a multiplicity of infection (MOI) of 20 for 2.5 h. Uninfected wells were treated with HBSS. The monolayers were washed and placed into 24-well plates. 600 μl of HBSS was added to the lower (apical) chamber and 100 μl of PMNs (1 × 106) was added to the top (basolateral) chamber. PMNs that transmigrated into the apical chamber were collected and lysed in 10% Triton-X 100. Migrated neutrophils were quantitated using myeloperoxidase ELISA following a well-established assay (McCormick et al., 1995 (link)) where serial dilutions of known numbers of neutrophils were used to establish a standard curve. For each donor the average migration from triplicate wells per condition was assessed and plotted.
5
Culturing Human Lung Cancer Cells
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Human pulmonary mucoepidermoid carcinoma-derived NCI-H292 (H292) cells were grown on the underside of collagen-coated Transwell filters (0.33-cm2, Corning Life Sciences) in RPMI 1640 medium (ATCC) with 2 mM L-glutamine, 10% FBS, and 100 U penicillin/streptomycin following a previously described protocol [9 (link)].
6
Transepithelial Barrier Integrity Assays
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Transepithelial electrical resistance (TEER) of HT-29cf8, and SK-CO15 intestinal epithelial cell monolayers cultured on collagen-coated transwell filters (Corning Incorporated) was measured using an EVOM2 Volt Ohm Meter (World Precision Instruments, Sarasota, FL). The resistance of cell-free collagen-coated filters was subtracted from each experimental point. A transmonolayer dextran flux assay was performed as previously described 41, 42 . IEC monolayers cultured on transwell filters were apically exposed to 1 mg/ml of FITC-labeled dextran (4,000 Da) in HEPES-buffered Hanks' balanced salt solution (HBSS). After 120 min of incubation, samples were collected from the lower chamber and FITC fluorescence intensity was measured using a SpectraMax M2 plate reader (Molecular Devices, San Jose, CA), at excitation and emission wavelengths 485 nm and 544 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.10.467974 doi: bioRxiv preprint nm, respectively. The amount of FITC-dextran translocated across the epithelial cell monolayer was calculated based on a calibration curve using Prism 9.10 software (GraphPad, La Jolla, CA).
The copyright holder for this preprint this version posted November 11, 2021. ; https://doi.org/10.1101/2021.11.10.467974 doi: bioRxiv preprint nm, respectively. The amount of FITC-dextran translocated across the epithelial cell monolayer was calculated based on a calibration curve using Prism 9.10 software (GraphPad, La Jolla, CA).
7
Podocyte Permeability Barrier Assay
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BSA permeability assay was used to evaluate the filtration barrier function of podocyte monolayers. Briefly, podocytes(5*10 3 ) were seeded onto collagen-coated trans-well filters( pore size, 3um; Corning,New York,NY,USA) and cultured under differentiation conditions. Cells were washed twice with PBS. Then, the top chamber was refilled with 0.2ml of RPMI 1640 medium and the bottom chamber with 1ml of RPMI 1640 medium containing 10ug/ml FITC-BSA, and incubated at 37°C. After 4 hours of incubation, a small amount of medium from the top chamber was collected and the fluorescence of FITC-BSA determined.
8
Albumin Permeability Assay in Podocytes
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Human podocytes were placed on a 12-well plate with collagen-coated Transwell filters (Corning) and differentiated at non-permissive conditions as described above by culturing for 10 days. The cells were then treated with CML-KLH (1 µg/ml) for 48 h. Subsequently, the medium was removed, and cells were washed twice with 1 mM CaCl 2 and 1 mM MgCl 2 mixture to preserve cadherin junctions. 2 ml of RPMI 1640 medium with 40 mg/ml BSA was then placed in the bottom chamber, and 0.3 ml of RPMI 1640 medium (without albumin) was placed in the top chamber. The cells were incubated at 37°C, aliquots of medium was collected from the top chamber at various (1, 2, and 4 h) time points, and albumin concentration was measured in these aliquots using the BCA protein assay kit (Sigma-Aldrich). Absorbance was measured at 562 nm using a Spectra max plate reader (Molecular Devices).
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