Two mm long human limbal segments were set up epithelial -side up on the center of 0.4-μm-pore 25-mm-diameter polyester membrane inserts (Corning Costar, Corning, NY). The inserts were pre-equilibrated in a supplemented hormonal epithelial medium (SHEM) consisting of 950 ml of a 1:1 mix of Dulbecco’s modified minimal essential medium and Ham F12 (DMEM/F-12, Gibco, Grand Island, NY), 50 ml of fetal bovine serum (FBS, Gibco), 5 ng of human recombinant epidermal growth factor, 28 mg of phosphoethanolamine, 1x ITS (Gibco) and 1x penicillin-streptomycin (Gibco) mixes alone (control medium) or complemented with 10 µM IWP2 (Tocris, Bristol, UK) or 3 µM CHIR99021 (Stemgent, San Diego, CA). A single batch of FBS was used in all experiments. Culture media were refreshed every 72 h. When outgrowths in control medium reached 70–80% confluence (typically in 10–14 days), outgrown cells were incubated in trypsin (Gibco) for 10 min to obtain fully dissociated cells. Cells were pelleted, resuspended in SHEM, and counted in a hemocytometer. Some cultures were washed with PBS, fixed-stained in 10% acetic acid–45% methanol containing 0.45% Commassie blue R 250, and photographed. Whenever a source is not indicated for a chemical, it was purchased from Sigma.
Polyester membrane insert
Manufactured by Corning
Sourced in United States
Polyester membrane inserts are a type of lab equipment used for cell culture applications. The inserts feature a porous polyester membrane that allows for the exchange of media, nutrients, and waste between the upper and lower chambers. The primary function of these inserts is to provide a platform for cell growth and experimentation.
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Lab products found in correlation
Matrigel invasion chamber, by Corning (2 mentions)
Diff quik staining kit, by Sysmex (2 mentions)
Millicell ers epithelial volt ohmmeter, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Fitc bsa, by Merck Group (1 mentions)
Spectramax i3x, by Molecular Devices (1 mentions)
Jem 1400 electron microscope, by JEOL (1 mentions)
Infinite f200 pro microplate reader, by Tecan (1 mentions)
Infinite f200 pro, by Tecan (1 mentions)
Evom2 meter, by World Precision Instruments (1 mentions)
Ham s f 12 dmem, by Merck Group (1 mentions)
A549 cells, by Cytion (1 mentions)
Htb 37, by LGC (1 mentions)
Glomax multi microplate multimode reader, by Promega (1 mentions)
Mem non essential amino acid, by Corning (1 mentions)
Mem vitamins, by Corning (1 mentions)
Pen strep, by Corning (1 mentions)
19 protocols using polyester membrane insert
1
Limbal Epithelial Cell Culture Protocol
2
Measuring Permeability of HGF Monolayers
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To determine the changes in permeability across HGF monolayers, HGFs were seeded at a density of 1.0 × 105 cells/well on top of 24-well transwell chambers with 6.5 mM diameter polyester membrane inserts (0.4 μM pore size, COSTAR, USA) and grown to confluence. HGFs were cultured in medium containing 1% FBS for 24 h until a well-formed monolayer was observed. HGFs were then incubated with TNF-α (10 ng/ml) and IL-1β (2 ng/ml) with increasing concentrations of CORM-3 for 24 h. Unstimulated cells were used as a control. At the end of stimulation, the treatment medium was carefully removed from each plate well, and FITC-BSA (10 mg/ml, Sigma, USA) and equimolar amounts of unlabeled BSA were added to the top and bottom chambers with phenol red-free DMEM for 2 h at 37°C in the dark. The medium from different chamber wells was then transferred to a blank 96-well opaque plate for fluorescence measurement. Fluorescence intensity was quantified at 494 nm excitation and 520 nm emission (SpectraMax i3X, Molecular Devices, USA).
3
Cell Invasion Assay Protocol
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Cell invasion assays were performed using 24-well Matrigel invasion chambers (8.0 μm pore size; Corning Inc., Corning, NY, USA) and polyester membrane inserts (8.0 μm pore size; Corning Inc.) according to the manufacturer’s instructions. Briefly, 2×104 cells in FBS-free medium were seeded in the upper chambers, and medium containing 10% FBS was added to the lower chambers. After 48 hours, the cells in the upper chamber were gently cleared with a cotton swab, and Diff-Quik staining kit (Sysmex, Hyogo, Japan) was used to stain the membranes to allow cell counting.
4
Cell Invasion and Migration Assay
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Invasion and migration assays were performed in 24‐well Matrigel invasion chambers (8.0 μm pore size; Corning, New York, NY) and polyester membrane inserts (8.0 μm pore size; Corning) according to the manufacturer's instructions. Briefly, medium containing 10% FBS was added to lower chambers and 5 × 104 cells in FBS‐free medium were seeded into upper chambers. After 48 h, cells on the upper chamber were removed with a cotton swab. Membranes were stained, using the Diff‐Quik staining kit (Sysmex, Hyogo, Japan) and cells were counted.
5
Visualizing Gold Nanoparticle Uptake in Cancer Cells
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To visualize the uptake of gold nanoparticles by cancer cells, transmission electron microscope (TEM) images were taken. For this 105 cells were seeded onto 0.4 µm pore size polyester membrane inserts (Corning, Corning, NY, USA). After cells were attached to the membrane, the cultures were incubated with AuNPs in 6.8 µM concentration for 24 h, then cells were washed with Phosphate-Buffered Saline (PBS) and fixed in 4% glutaraldehyde. Samples were first embedded in gelatin, then were sliced into 1–2 mm cubes which were subsequently embedded in epoxy (Embed 812, EMS, PA 19440). To identify the cell monolayer, semi-thin sections of 1 µm were prepared, then thin sections of 70 nm were obtained and stained with uranyl and lead solutions. Images were captured by a Jeol JEM-1400 electron microscope (Jeol Ltd., Tokyo, Japan) using 100 kV voltage.
6
Measuring Epithelial Barrier Function
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Cells were cultured on transwell plates (0.4 μm pore size, 12 mm diameter) with polyester membrane inserts (Corning Incorporated, Corning, NY, USA). The barrier function of TJs was estimated by TER and paracellular flux of 2-NBDG. TER was measured using volt ohmmeter [33 (link),35 (link)]. In the 2-NBDG flux assay, 100 μM 2-NBDG was added to transwell inserts. After incubation at 4 °C for 30 or 60 min, the solution in the lower compartment was collected and the fluorescence intensity of 2-NBDG was measured using an Infinite F200 PRO microplate reader (Tecan, Mannedorf, Switzerland). The concentration of 2-NBDG was calculated using a calibration curve.
7
Transwell-based Permeability Assay
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A549 cells were cultured on transwell plates (0.4 μm pore size, 12 mm diameter) with polyester membrane inserts (Corning Incorporated, Corning, NY, USA). The barrier function of TJ was estimated by TER as described previously [42 (link)]. An apparent permeability coefficient (Papp) of DXR and LY was calculated as the following equation.
whereby dC/dt is the change in basal concentration in 30 min (μg/mL/s), V is the volume of the chamber, C0 is the initial concentration of DXR and LY in the apical solution, and A is the area of the chambers (cm2).
whereby dC/dt is the change in basal concentration in 30 min (μg/mL/s), V is the volume of the chamber, C0 is the initial concentration of DXR and LY in the apical solution, and A is the area of the chambers (cm2).
8
Lymphocyte Migration Assay Protocol
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For evaluating lymphocyte migration, experiments were performed in 24-well plates with 5 μm-pore size polyester membrane inserts (Corning Inc.). rhIL-2-activated PBL (105) in 200 μl of RPMI medium were placed in the upper chamber. NHA cells (3 × 105/300 μl) under different treatments for 24 h were seeded on lower chambers. Lower chambers with medium only served as a control for spontaneous migration. After 4 h incubation at 37 °C migrated cells were counted and stained with anti-CD3-PE, anti-CD19-ECD, anti-CD56-PC5, anti-CD4-PC7 and anti-CD8-FITC. The number of migrated T, B and NK cells was counted and expressed as a percentage of the total migrated cells.
9
Macrophage-Mesenchymal Stem Cell Coculture
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In setup B, the cocultures of the MPs with the MSCs (1:4 MP:MSC) and BCP 60/40 or BCP 20/80 granules were set up via transwell assays using polyester membrane inserts with a 0.4 μm pore size (Corning, Lowell, MA, USA); transwell membranes allow cellular interactions without direct cell-to-cell contact. The MPs cocultured with the monolayer MSCs served as controls. The MSCs were seeded on the BCP granules in notched 24-well plates as described above. Separately, the U937 cells were seeded in transwell inserts and stimulated with PMA for 48 h to induce MP differentiation, and they were allowed to mature in GM for an additional 24 h. Thereafter, the inserts with adherent MPs were transferred to the notched wells with the MSCs, and the coculture was initiated in GM for an additional 72 h. In relevant groups, the culture media were supplemented with cytokines to simulate an inflammatory microenvironment.
10
Transepithelial Electrical Resistance and Permeability Assay
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Cells were cultured on transwells with polyester membrane inserts (Corning Incorporated-Life Sciences, Acton, MA). TER was measured using a Millicell-ERS epithelial volt-ohmmeter (Millipore, Billerica, MA, USA). TER values (ohms × cm2) were normalized by the area of the monolayer and were calculated by subtracting the blank values from the filter and the bathing medium. The paracellular diffusion of LY for 1 h from the apical-to-basal compartments was measured with an Infinite F200 pro (Tecan). PCl/PNa was measured by Ussing chamber assay. Both apical and basal chambers were filled with Hank’s Balanced Salt Solution (HBSS). To measure dilution potential, the basal HBSS was replaced with HBSS containing half NaCl concentration in which osmolarity was balanced with mannitol. The absolute permeability values, PNa and Cl−, were calculated as described elsewhere [29 (link)].
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