An indirect coculture system was established with transwells with a 0.4 µm pore polyester membrane insert (Corning, USA). Activated T lymphocyte-dominated PBMCs were incubated with GC cells and organoids in 6-well and 12-well plates, respectively. After 48 h of incubation, the PBMCs were collected and stained for flow cytometry.
0.4 m pore polyester membrane insert
Manufactured by Corning
Sourced in United States
The 0.4 µm pore polyester membrane insert is a laboratory equipment product. It is designed to filter and separate materials based on their size. The core function of this product is to allow the passage of particles smaller than 0.4 micrometers while retaining larger particles.
Automatically generated - may contain errors
Lab products found in correlation
Pneumacult ali basal medium, by STEMCELL (2 mentions)
Fitc conjugated dextran, by Tokyo Chemical Industry (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Cryostor cell cryopreservation medium, by Merck Group (1 mentions)
Epon 812, by Merck Group (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Rna solv reagent, by Omega Bio-Tek (1 mentions)
8 protocols using 0.4 m pore polyester membrane insert
1
Indirect co-culture of T cells and GC cells
2
Lipid-mediated Endothelial Permeability
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EA.hy 926 cells were seeded into a transwell chamber with a 0.4 µm pore polyester membrane insert (Corning) at a density of 1 × 105 cells/chamber two days before the treatment. After incubation with the indicated lipids for 1 h, H2O2 (500 µM)-induced permeability was quantified using FITC-conjugated dextran (10 kDa, 1 mg/mL, Tokyo Chemical Industry Co., Ltd., JP) for 2 h. The fluorescence intensity in the lower chambers was determined using a fluorescence reader (EnSpire Multimode Plate Reader, PerkinElmer, Waltham, MA, USA).
3
Cytokine Secretion of Macrophages Exposed to Alloys
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To conduct these experiments, THP-1 cells differentiated into macrophages were used. A previously sterilized alloy sample was placed on a 6.5 mm Transwell® with a 0.4 µm Pore Polyester Membrane Insert (Corning, Corning, NY, USA) placed over the macrophage cultures (cell culture insert system) and maintained in close contact with the cells for 24 h to analyze the secretion of inflammatory cytokines. As a positive control, 1 µg/mL of lipopolysaccharide (LPS) (Sigma-Aldrich) was added to the macrophages culture. As a negative control, macrophages were cultured in the absence of the alloys or LPS. After 24 h, supernatants were collected and used to quantify cytokine secretion. Inflammatory cytokines TNF-α, IL-1β and IL-6 were evaluated by flow cytometry using cytometric bead array (CBA) (Becton–Dickinson, East Rutherford, NJ, USA). Cytokine concentrations in the supernatant were analyzed according to the manufacturer’s protocol. Negative control was considered as basal secretion level with a 0 value. Experiments were performed in triplicate.
4
Cryopreservation of Diagnostic PCD Cells
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Between March 2018 and August 2020 UHS has cryopreserved surplus diagnostic cells from 181 PCD clinic patient samples and 30 healthy donor samples. Surplus cells from passage 1 were frozen 1 million per cryovial in 1 mL CryoStor® cell cryopreservation medium (Sigma, St. Louis, MO, USA, #C2874). Cells were initially frozen at −80 °C (graduated freezing −1 °C/minute in a Mr. FrostyTM container Thermo Fisher Scientific, Waltham, MA, USA, #5100–0001), then transferred to liquid nitrogen for longer-term storage. After thawing, washed cells were seeded for research in a smaller Transwell® insert format in 24-well plates. Briefly, 50,000 cells per collagen-coated 6.5 mm Transwell® with 0.4 µm pore polyester membrane insert (Corning Life Sciences, Corning, NY, USA, #3470) in 100 µL PneumaCult Ex plus medium supplemented (apical side) and 350 µL of the same medium on the basolateral side. Cultures were taken to ALI after 1–2 days replacing only the basolateral medium with 350 µL PneumaCult ALI medium supplemented and maintained as detailed above.
5
Visualizing TiO2 NP Uptake in A549 Cells
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To detect intracellular accumulation of TiO2 NPs in A549 cells, 105 cells were seeded onto 0.4 µm-pore polyester-membrane inserts (Corning, Corning, NY, USA) placed in a six-well plate. On the following day, cells were exposed to 0.1 mg/mL TiO2 NPs for 24 hours. Treated cells were washed in PBS, fixed in 4% glutaraldehyde diluted in PBS for 2 hours, and subsequently embedded in gelatin (2% gelatin in PBS). The specimen obtained was sliced to 1–2 mm cubes, which were further embedded in epoxy (Epon 812; Sigma-Aldrich) by a routine TEM sample-preparation protocol. Semithin sections of 1 µm were prepared to identify the cell monolayer. Blocks were trimmed and thin sections of 70 nm obtained and stained with uranyl and lead solutions. Images were captured with a JEOL 1,400 plus 120 kV using 100 kV voltage.
6
Differentiation of NHBE Cells at Air-Liquid Interface
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Transwells (6.5 mm) with 0.4 µm-pore polyester membrane inserts (Corning Inc.) were coated with PureCol for 20 min before cell seeding. NHBE cells (5 × 104) suspended in 200 µl of complete AEC medium were seeded in the apical part of each Transwell. Then, 500 µl of complete AEC medium was added to the basal part of the Transwell. When the cells formed a confluent layer on the Transwell insert, the AEC medium was removed from the apical insert, and PneumaCult-ALI basal medium (Stemcell Technologies) with the required supplements (Stemcell Technologies), 2% penicillin/streptomycin and 1% amphotericin B was added to the basal chamber. The ALI medium in the basal chamber was changed every other day. The apical surface was washed with 1x Dulbecco's phosphate-buffered saline (DPBS) (Thermo Fisher Scientific) once per week initially but more frequently when more mucus was observed on later days (when it was difficult to see the apical cells, it was determined that the difficulty was probably due to a thick layer of mucus). All cells were differentiated for up to four weeks (at 37 °C with 5% CO2) until the desired cellular and physiological properties of an epithelial layer were obtained, such as a CBF greater than 6 Hz and a TEER greater than 500 ohm/cm2.
7
SARS-CoV-2 Infection Dynamics in Nasal Epithelial Cells
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Differentiated primary nasal epithelial cells seeded onto 0.4 µm pore polyester membrane inserts (Corning, Corning, NY, USA) were inoculated with SARS-CoV-2 BavPat1 isolate at an MOI of 10 at both the apical and basolateral surfaces. At 2 hpi, the inoculum was discarded, cells were washed with PBS, and fresh PneumaCult-ALI Basal Medium (Stemcell) without heparin, hydrocortisone, and antibiotics was added to the basolateral compartment. At the desired time points, 200 µL of pre-warmed medium was added to the apical surface of the cells and then incubated for 10 min. at 37 °C. Apical and basolateral supernatants were collected for titration or RNA isolation. Intracellular RNA was isolated using RNA-Solv reagent (Omega Bio-Tek, Norcross, GA) according to the manufacturer’s protocols, while using glycogen during precipitation.
8
Transwell Assay for Tight Junction Evaluation
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Cells were cultured on Transwell plates with 0.4 µm pore polyester membrane inserts (Corning Incorporated, Corning, NY, USA). The barrier function of TJs was examined by measuring TER and fluxes for LY (MW: 457) and DXR (MW: 580) as described previously [44 (link),45 (link)].
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