Migration assays were performed using 3.0-μm transwell Permeable Supports (CoStar). 0.1 × 106 IL-7-expanded TCRγδ cells, stimulated or not with IL-23 10 ng/mL overnight, were placed in transwell inserts in duplicates in 100 μL of media (IMDM-10%-2ME + 5ng/mL IL-7), in presence or absence of IL-23 (10 ng/mL). The lower chamber was filled with media alone, IL-23, or IL-23 plus the indicated pharmacological inhibitors. For conventional chemotactic assays, 0.1 × 106 IL-7-expanded TCRγδ cells well placed in transwell inserts in duplicates in 100 μL of media, and the lower chamber was filled with 600 μL of media alone, CCL2 (100 ng/mL; Prepotec) or CCL20 (500 ng/mL; CCL20). The number of migrated cells in the lower chamber was determined after 6 h by flow cytometry. For migration assays with sorted nTh17 and iTh17 cells, 0.1 × 106 cells were placed in transwell inserts and 10 μL of AccuCheck Counting Beads (ThermoFisher) were added to the bottom chamber; 2 h later, cells in bottom chamber were collected and stained with CD4 and CD44 before flow cytometry analysis. The migration index was obtained normalizing number of cells in lower chamber to the untreated condition.
Permeable support
Manufactured by Corning
Sourced in United States
Permeable supports are a type of laboratory equipment used to facilitate the movement of liquids, gases, or small particles across a semi-permeable membrane. They are designed to allow the passage of specific substances while retaining others, depending on the specific application.
Automatically generated - may contain errors
Lab products found in correlation
Differential quik stain kit, by Polysciences (2 mentions)
Matrigel basement membrane matrix, by Corning (2 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Cf inhibitor 172, by Merck Group (1 mentions)
Fitc conjugated dextran, by Merck Group (1 mentions)
24 well companion plate, by Corning (1 mentions)
Keratinocyte serum free media, by Thermo Fisher Scientific (1 mentions)
Cytoselect 24 well cell invasion kit, by Cell Biolabs (1 mentions)
Digital sight ds fi1 camera, by Nikon (1 mentions)
Basement membrane extract, by Bio-Techne (1 mentions)
Insulin transferrin selenium supplement, by Thermo Fisher Scientific (1 mentions)
Pneumacult ali 10x supplement, by STEMCELL (1 mentions)
L glutamine 200 mm, by Capricorn (1 mentions)
Hepes 1 m, by Capricorn (1 mentions)
Pneumacult ali maintenance supplement, by STEMCELL (1 mentions)
Hydrocortisone, by STEMCELL (1 mentions)
Fetal bovine serum (fbs), by Capricorn (1 mentions)
Bronchial epithelial growth media, by Lonza (1 mentions)
Dvc 1000 voltage clamp, by World Precision Instruments (1 mentions)
Millicell ers voltammeter, by Merck Group (1 mentions)
14 protocols using permeable support
1
TCRγδ Cell Migration Assay
2
CFTR Function Assessed in Airway Epithelial Cells
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CFBE41o-cells expressing WT or F508del CFTR were seeded onto permeable supports (Costar) after coating with fibronectin [24 (link)]. Cells were grown to confluence and transferred to an air—liquid interface, after which they were mounted in modified Ussing chambers. Monolayers were initially bathed on both sides with identical Ringers solution containing (in mM): 115 NaCl, 25 NaHCO3, 2.4 KH2PO4, 1.24 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, 10 D-glucose (pH 7.4) and vigorously stirred and gassed with 95%O2: 5% CO2 at 37°C. Short-circuit current (Isc) was obtained using an epithelial voltage clamp. The mucosal bathing solution was changed to a low Cl- solution containing (in mM): 1.2 NaCl, 115 Na gluconate, plus 100 μM amiloride followed by addition of agonists (20 μM forskolin, 50 μM genistein, and/or 10 μM ivacaftor) to the mucosal surface. CF inhibitor 172 (10 μM; Sigma-Aldrich, St. Louis, MO) was added to the bathing solution at the conclusion of each experiment to block CFTR-dependent Isc.
3
Endothelial Barrier Disruption Assay
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LMVEC were seeded on permeable supports (3 μm pore size, Corning) placed into a 24-well plate, and grown to confluence. Cells were treated with or without HA purified from patient plasma samples (750 ng HA) or 1.5% patient plasma for 16 hours at 37°C to induce endothelial barrier disruption. The upper chamber was replaced with FITC-conjugated dextran (1 mg/mL, 40 kDa, MilliporeSigma) in PBS, and a sample of medium from the lower chamber was measured after 1 hour.
4
Cell Invasion Assay using Matrigel
5
Esophageal Epithelial Differentiation
6
Cell Invasion Assay with rHE4
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For one replicate of the invasion assays, a Cytoselect 24-Well Cell Invasion Kit (8 μm, Colorimetric, Cell Biolabs, CBA-110) was used according to the manufacturer’s instructions. After overnight starvation, OVCAR8 cells (1 × 105/well) were plated in triplicate in serum-free media in cell culture inserts in the presence or absence of 20 nM rHE4. Media containing 10% FBS were inserted into the lower chamber. After 24 h, media were aspirated from the insert, and the top side of the insert was cleaned with a cotton swab. The insert was then crystal violet stained and washed, and the stained cells were extracted. Extraction solution (150 µL) from each sample was then transferred to a 96-well plate and OD measured at 550 nm. For the following two experimental replicates, 8 µm Transwell Permeable Supports Coated with Cultrex BME (Corning Inc., 3458) were used, with crystal violet staining and acetic acid extraction.
7
Transwell Invasion and Migration Assays
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The transwell assays with matrigel was used for cell invasion assays. Permeable supports (Corning Incorporated Life Sciences, USA) were precoated with 40 ul of 1 mg/ml matrigel. Then freshly isolated BMCs 4×10 6 cells suspended in 200 ul medium without FBS were placed on the top chamber of each well (Millipore, MA, USA). For migration assays, freshly isolated BMCs 5×10 6 and 4×10 4 4T1 cells suspended in 200 ul medium without FBS were placed on the upper chamber without matrigel (Millipore, MA, USA). The lower chamber was lled with 800 ul of medium with 10% FBS as the nutritional attractant.
Then the cells were xed with 4% polyformaldehyde for 1 h and then stained with 0.1% crystal violet.
Cleaned off the cells that not invaded to the lower surface of membrane. The membranes were then visualized under an optical microscope at 200 ×. Images were captured with a Nikon Digital Sight DS-Fi1 camera and NIS-Element software. Cells in three different visual elds were counted.
Then the cells were xed with 4% polyformaldehyde for 1 h and then stained with 0.1% crystal violet.
Cleaned off the cells that not invaded to the lower surface of membrane. The membranes were then visualized under an optical microscope at 200 ×. Images were captured with a Nikon Digital Sight DS-Fi1 camera and NIS-Element software. Cells in three different visual elds were counted.
8
Isolation and Culture of Rat TAL Cells
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Individual TAL cells were obtained from rat TAL suspensions after digesting the tubules with collagenase (0.1%), trypsin (0.25%), and DNase (0.0021%). Single TAL cells were isolated by centrifugation at 41,300 × g in a 35% Percoll gradient and seeded in permeable supports (Corning Inc.) coated with basement membrane extract (Trevigen, Gaithersburg, MD). Cells were grown in DMEM supplemented with 1% fetal bovine serum and insulin-transferrin-selenium supplement (Invitrogen) at 37 °C and 5% CO2.
9
Culturing Airway Epithelial Cells at ALI
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All cells were grown at 37 °C in a humidified atmosphere with 5% (v/v) CO2. Culture conditions for CFBE and HT29 cells have been described earlier [25 (link)]. In brief, airway epithelial cells were grown in DMEM/Ham’s F-12 with L-Glutamine medium supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) L-glutamine 200mM and 1% (v/v) HEPES 1M (all from Capricorn Scientific, Ebsdorfergrund, Germany). CFBE parental cells were grown in MEM with Earle’s Salts with L-Glutamine medium (Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% FBS. The airway epithelial cell line H441 was grown in RPMI and DMEM media. The immortalized human airway basal cell line BCi-NS1 (kindly provided by Prof. Ron Crystal, Weill Cornell Medical College, New York City, NY, USA) was maintained in Bronchial Epithelial Growth Media (Lonza). Cells were differentiated by growing on permeable supports (Snapwell, Corning, NY, USA) in an air-liquid interface (ALI) for up to 30 days in PnemaCult-ALI medium supplemented with PneumaCult-ALI 10X Supplement, PneumaCult-ALI Maintenance Supplement, hydrocortisone and heparin (all from StemCell Technologies, Vancouver, BC, Canada), and 1% penicilin-steptomycin (10 000 U/mL; Gibco, ThermoFisherScientific, Waltham, MA, USA).
10
Cell Migration Assay Protocol
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Cells (106 cells/100 μl) in serum-free DMEM were added to the upper chamber and 500 μl of the DMEM with 10% FBS was added to the lower chamber with permeable supports (Corning, NY, USA). Then, cells on the upper surface which were incubated for 24 hours at 37°C were removed using a cotton-tipped applicator. Finally, cells on the lower surface of the filter were stained with crystal violet to calculate the average number of migrated cells [25 (link)].
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