The inserts for 24-well plates (transparent PET membrane, 8.0 μm pore size, Falcon) were coated with Geltrex Matrix (Invitrogen). 30.000 Panc-1 cells with aphidicolin (5 μM) and with/without AZ10606120 (10 μM), BzATP (10 μM) and ATP (100 μM) were placed in the upper chamber in 1 % of serum, and media with 10 % of serum was added in the lower chamber. After 24 h incubation at 37 °C in a humidified atmosphere with 5 % CO2, cells were fixed in cold methanol and stained with Crystal Violet. Bright field images were taken with 10x objective in Leica DMI6000B microscope. Cells were counted by ImageJ, NIH.
Transparent pet membrane
Manufactured by BD
Sourced in Germany
Transparent PET Membrane is a laboratory equipment product made of polyethylene terephthalate (PET) material. It is a thin, transparent membrane used for various laboratory applications that require a semi-permeable barrier.
Automatically generated - may contain errors
Lab products found in correlation
Geltrex matrix, by Thermo Fisher Scientific (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Cell strainer, by Corning (1 mentions)
Giemsa stain solution, by Fujifilm (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Tc20 automated cell counter, by Bio-Rad (1 mentions)
Rpmi 1640, by American Type Culture Collection (1 mentions)
Non essential amino acids (neaa), by Harvard Bioscience (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Penicillin streptomycin, by GE Healthcare (1 mentions)
Dulbecco s modified eagle s medium high glucose, by GE Healthcare (1 mentions)
Caco 2, by GE Healthcare (1 mentions)
Transwell insert, by BD (1 mentions)
Adamts4 sirna, by Horizon Discovery (1 mentions)
Spectramax m2 m2e multimode plate reader, by Molecular Devices (1 mentions)
6 protocols using transparent pet membrane
1
Panc-1 Cell Migration Assay
2
Transwell Assay for Cell Migration
3
Migration and Invasion Assays of Cells
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Cells were dissociated with trypsin (Gibco) to form a single cell suspension using a 40-μm Corning® Cell Strainer (Corning, Inc., Corning, NY, USA) and counted with a TC20™ Automated Cell Counter (Bio-Rad Laboratories). Migration assays were performed using Falcon® Cell Culture Inserts and Companion Plates in a 24-well plate with an 8.0-µm Transparent PET Membrane (Falcon, Corning, Inc.). Cells (5 × 105) were suspended in 500 μL of serum-free medium and seeded into the upper chamber with or without 10 µM Y-27632 (Selleck Chemicals). The lower chamber was filled with a medium containing 20% fetal bovine serum. After incubation for 24 h at 37°C, in an atmosphere containing 5% CO2, migrated cells were fixed in 100% methanol for 10 min and stained with Giemsa Stain Solution (FUJIFILM Wako) for 30 min. The migrated cells were counted in five random fields per sample under a microscope at 200× magnification (Olympus Corporation, Tokyo, Japan). Invasion assays were performed using the same method as that used for the migration assays, with the following exceptions: 1 × 105 cells were seeded into the upper chamber of a Matrigel-coated transwell chamber (Corning® BioCoat™ Matrigel® Invasion Chambers 24 Well, Corning, Inc.), and invaded cells were counted after incubation for 48 h.
4
Co-culture of Macrophages and Alveolar Cells
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For co-culture experiments, primary macrophages isolated from mice fed standard diet, control diet, or DHA supplemented diet were plated in six well co-culture inserts (Falcon’s Transparent PET Membrane, 2.0 μm pore size, 1 × 105 pores/cm2). The macrophages cultured in the transwell inserts were treated as described above, washed with fresh media, and placed over wells which contained confluent MLE12 cells (1 × 105 cells/well) in HITES media, described elsewhere40 (link). The procedure was using an immortalized mouse alveolar macrophage cells line (MHS). MHS cells were cultured in RPMI-1640 (ATCC, Manassas, VA) under standard conditions. After 24 h of growth, the culture media was harvested from individual wells and the levels of HMGB1 and LTB4 were measured (MHS cytokine responses, Supplemental Figure 1 ). The MLE12 cells were stained and evaluated for apoptosis, cl-caspase 3, and proliferation, Ki67, by flow cytometry.
5
Caco-2 Cell Culture for Intestinal and Metabolic Studies
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The human epithelial cell line Caco2 (carcinoma colon-2 cell, American Type Culture Collection, Manassas, VA) is not only accepted as a model for intestinal barrier [36] (link) but for fructose metabolism [37] (link) as well. Caco2 cells, passages 37–47, were maintained in Dulbecco's modified Eagles' medium high glucose (4,5 g/l, PAA Laboratories, Pasching, Austria) supplemented with 20% fetal bovine serum (FBS) (Biochrom AG, Berlin Germany), 1% non-essential amino acids (Biochrom AG, Berlin Germany) and 1% penicillin-streptomycin (PAA Laboratories, Pasching, Austria) in a humidified 5% CO2 atmosphere at 37°. Cells were fed every 2 or 3 days and transferred after reaching 75% of confluence to transwell systems (transparent PET Membrane, 0.4 µm pore size, 4.2 cm2 growth area; BD Falcon, Heidelberg, Germany) at a density of 5*105 cells per well.
After 13 days cells differentiated completely and were maintained with DMEM supplemented with 1% NEA and 1% penicillin-streptomycin, without FBS 24 h.
After 13 days cells differentiated completely and were maintained with DMEM supplemented with 1% NEA and 1% penicillin-streptomycin, without FBS 24 h.
6
Endothelial Barrier Permeability Assay
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Boyden chambers were used for permeability evaluation; HMVEC-Ls were seeded on Transwell Inserts (1.0 µm pore; Falcon ® Transparent PET Membrane) at a concentration of 1 × 10 5 cells/well. The cells were cultured for 2 days. After monolayer confluency was confirmed, control or ADAMTS4 siRNA (Horizon Discovery Ltd., Waterbeach, UK) was transfected. After overnight culturing, cells were stimulated with or without LPS (10 ng/mL) for 24 h. To measure dextran leakage in human HMVEC-Ls, 100 µg/mL of assay solution containing FITC-labeled dextran (Molecular weight 3000, MERCK, Darmstadt, Germany) was added to each upper chamber, and after 20 min of incubation at 37 • C, the fluorescence intensity of the medium from the lower chamber was measured at 485-538 nm using a SpectraMax M2/M2e multi-mode plate reader (Molecular Devices, San Jose, CA, USA).
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