For cell characterization, haRPE cells (4 × 104 cells/well) were seeded onto 6.5-mm transwell inserts (Corning; Tewksberg, MA, USA). After culturing for 5 weeks, samples were collected from apical and basal chambers 24 h after media change. For inflammasome experiments, media from treated and untreated cells was collected from 6-well plates 24 h after activation. ELISAs for Pigment Epithelium-Derived Factor (PEDF) (R & D Systems; Minneapolis, MN, USA), Vascular Endothelial Growth Factor-A (VEGF-A) (eBioscience; San Diego, CA, USA), Interleukin-1β (IL-1β) (eBioScience), IL-6 (BD Bioscience; San Jose, CA, USA), and C3a (BD Bioscience) were conducted according to the manufacturer’s protocols. Concentrations of protein were derived from a standard curve and normalized to chamber volume.
6.5 mm transwell insert
Manufactured by Corning
Sourced in United States
The 6.5-mm Transwell inserts are a laboratory equipment used for cell culture applications. They provide a porous membrane support to facilitate the study of cellular interactions, migration, and barrier function.
Automatically generated - may contain errors
Lab products found in correlation
Vascular endothelial growth factor a vegf a, by Thermo Fisher Scientific (2 mentions)
Axiovert 25, by Zeiss (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Interleukin 1β il 1β, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by BD (1 mentions)
Human intesticult organoid growth medium, by STEMCELL (1 mentions)
Millicell ers 2 voltohmmeter, by Merck Group (1 mentions)
Cangrelor, by Bio-Techne (1 mentions)
2 mesadp, by Merck Group (1 mentions)
Adenosine diphosphate (adp), by Merck Group (1 mentions)
Psb0739, by Bio-Techne (1 mentions)
2 mesadp, by Bio-Techne (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
14 protocols using 6.5 mm transwell insert
1
Characterization of Human RPE Cells
2
Seawater-based Nasal Hygiene Protocol
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Stérimar Nasal Hygiene (SNH, Laboratoire Fumouze, Levallois-Perret, France) contains 31.82 ml seawater and purified water (qsp 100 ml) and is sterilized by microfiltration. Assays were performed in a 3D reconstituted human nasal epithelium model, MucilAir™ (Epithelix Sàrl, Geneva, Switzerland), a mixture of human nasal cells isolated from a panel of 14 different donors. Cells were cultured in 500 µl of MucilAir™ culture medium in a CO2 incubator (37 °C, 5% CO2, 100% humidity, Heracell, Waltham, Massachusetts, United States) in 24-well plates with 6.5-mm Transwell® inserts (Corning Life Sciences, Corning, New York, United States). Before treatments, inserts were washed with 200 µl of MucilAir™ culture medium and the quality of the tissue was assessed under an inverted microscope (Zeiss Axiovert 25, Oberkochen, Germany).
3
Swine Enteroid Culture and Characterization
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Swine enteroids were cultured, passaged, and plated on 6.5 mm transwell inserts (Corning, Ref: 3470), as described previously [32 (link)]. Cell growth was monitored until the membrane was covered by cells. Transepithelial resistance (TER) was measured with a Millicell-ERS2 Volt-Ohm meter (Millipore Sigma, St. Louis, MO, USA) before media change and the experiment was carried out when TER reached a minimum of 600 ohms/cm2 and was constant. Swine enteroids were maintained in Intesticult Organoid Growth Medium (Human; Stemcell Technologies, Vancouver, BC, Canada) which will be referred to as the culture media in this report.
4
Transwell Invasion Assay under Glucose Concentrations
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Transwell invasion assay was performed using 6.5 mm transwell inserts (Corning®, Glendale, AZ, USA) with 8.0 µm pore polycarbonate membrane. Inserts were treated
beforehand with MatrigelTM Matrix Basement Membrane (BD Biosciences, Franklin Lakes, NJ, USA) in coating buffer (250 µg/ml) and incubated for 2 h. B16-F10 cells
were cultured in serum-free media for 48 h and were collected by trypsin/EDTA treatment. Cells collected were suspended in serum-free media containing glucose concentrations of 11 mM, 33 mM,
and 55 mM. 5.0 × 104 cells were seeded in the upper chamber of the insert and complete R10 media were added in the lower chamber. After 22 h of incubation, the leftover cells in
the upper chamber were discarded using a cotton bud, and cells on the lower surface were fixed with 100% methanol and stained with hematoxylin and eosin solutions. Ten random fields of each
membrane were observed and photographed under a microscope with 4× magnification. Cells were then analyzed and counted.
beforehand with MatrigelTM Matrix Basement Membrane (BD Biosciences, Franklin Lakes, NJ, USA) in coating buffer (250 µg/ml) and incubated for 2 h. B16-F10 cells
were cultured in serum-free media for 48 h and were collected by trypsin/EDTA treatment. Cells collected were suspended in serum-free media containing glucose concentrations of 11 mM, 33 mM,
and 55 mM. 5.0 × 104 cells were seeded in the upper chamber of the insert and complete R10 media were added in the lower chamber. After 22 h of incubation, the leftover cells in
the upper chamber were discarded using a cotton bud, and cells on the lower surface were fixed with 100% methanol and stained with hematoxylin and eosin solutions. Ten random fields of each
membrane were observed and photographed under a microscope with 4× magnification. Cells were then analyzed and counted.
5
Infection Assay for Airway Epithelial Cells
6
Secreted Factors from iPSC-RPE Cells
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iPSC-RPE cells (4 × 104 cells/well) were seeded onto 6.5mm transwell inserts (Corning; Tewksburg, MA, USA) coated with Matrigel (Corning). After culturing for 5 weeks, samples were collected from apical and basal chambers 24hr after media change. ELISAs for Pigment Epithelium-Derived Factor (PEDF) (R & D Systems; Minneapolis, MN, USA), Vascular Endothelial Growth Factor-A (VEGF-A) (eBioscience; San Diego, CA, USA), Interleukin-6 (IL-6) (BD Biosciences; San Jose, CA, U.S.A), CFH (Abcam; Cambridge, United Kingdom) and C3a (BD Biosciences) were conducted according to the manufacturer’s protocols. Growth factor concentration was derived from a standard curve and normalized to chamber volume.
7
Transwell Bacterial Invasion Assay
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Cells were seeded in the bottom chamber of 6.5-mm Transwell inserts (#3470, Corning, Tewksbury, MA) at 8.0 × 10⁴ cells, and bacteria were seeded in the top chamber at a multiplicity of infection of 50 for the indicated time.
8
Respiratory Sensitizer Evaluation in 3D Nasal Model
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The remaining assays were performed in a 3D reconstituted human nasal epithelium model, MucilAir™ (Epithelix Sàrl, Geneva, Switzerland). Mucilair is a mixture of human nasal cells isolated from a panel of 14 different donors and has been selected for its great potential as a model to test respiratory sensitizers.13–16 (link) Cells were cultured in 500µL of MucilAir™ culture medium in a CO2 incubator (37°C, 5% CO2, 100% humidity, Heracell, Waltham, Massachusetts, United States) in 24-well plates with 6.5-mm Transwell® inserts (Corning Life Sciences, Corning, New York, United States). Before treatments, inserts were washed with 200µL of MucilAir™ culture medium and the quality of the tissue was assessed under an inverted microscope (Carl Zeiss Axiovert 25, Oberkochen, Germany).
9
Cytotrophoblast Cell Invasion Assay
10
Transwell Assay for Cell Migration and Invasion
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For migration assays, TNBC cells were transfected with miR-3189-3p or scramble control using Lipofectamine RNAiMAX. After 72 h, cells were detached from the plate and seeded into the 6.5 mm transwell insert (8.0 μm pore size; Corning, NY) at the final concentration of 3 × 104 cells per chamber in 200 μl of serum-free medium. 600 μl of complete culture medium supplemented with 10% FBS as chemoattractant were added to the lower chamber and cells were incubated at 37 °C. After 24 h, the inserts were gently washed 3 times with cold PBS and cells were fixed using 100% methanol. Non-migrated cells were removed from the top of the inserts using cotton swabs. After fixation, the cells migrated on the bottom of the insert were washed again with PBS and stained with 0.4% crystal violet. Migrated cells were counted from three random fields per insert at the magnification of 20× and averaged from at least three biological replicates. For the invasion assay we used Corning BioCoat Matrigel Invasion Chambers with 8.0 μm PET Membrane from Corning (Corning, NY). The protocol for invasion was essentially the same as the migration assay, except that we plated 8 × 104 MDA-MB-231 cells or 10 × 104 MDA-MB-468 cells per chamber in a total of 500 μl of serum-free medium, and 750 μl of complete culture medium containing 10% FBS as chemoattractant were added to the lower chamber.
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