The cellular potential for invasion was determined using a 24-well Transwell plate with 8.0-µm Pore Polyester Membrane Inserts (Corning, Inc.). Matrigel (Corning, Inc.) was melted overnight at 4°C and diluted to a final concentration of 1 mg/ml in pre-cooled serum-free medium. Then, 100 µl diluted Matrigel was added to the bottom of the upper chamber. The plate was then incubated at 37°C for 4–5 h to dry the Matrigel. At 24 h after transfection, HCT116 cells in the logarithmic growth phase were seeded in triplicate at a density of 1×106 cells/chamber. Cells were seeded on top of the Transwell plate in 100 µl DMEM supplemented with 0.1% bovine serum albumin (Thermo Fisher Scientific, Inc.). Then, 0.8 ml DMEM supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) was added to the lower chamber as a chemoattractant. After 24 h of incubation at 37°C, cells on the top surface of the insert were removed with a cotton swab. Cells that had invaded the lower surface of the membrane were fixed with 4% paraformaldehyde at 37°C for 15 min and stained with 800 µl Giemsa solution at 37°C for 20 min (Beyotime Institute of Biotechnology). Cells were visualized using a light microscope (CKX-41; Olympus Corporation). Cell counts were obtained from three randomly selected optical fields.
Pore polyester membrane insert
Manufactured by Corning
Sourced in United States
The Pore Polyester Membrane Insert is a lab equipment product designed for cell culture applications. It features a polyester membrane with pores of a specified size, providing a barrier for the controlled passage of cells, media, and other substances between the upper and lower chambers of a culture system.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Eclipse ts100, by Nikon (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Giemsa solution, by Beyotime (1 mentions)
Ckx41, by Olympus (1 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Image analysis system, by Leica (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Millicell ers voltohmmeter, by Merck Group (1 mentions)
11 protocols using pore polyester membrane insert
1
Transwell Invasion Assay for HCT116 Cells
2
Evaluating siRNA RTN Mucus Penetration
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Example 7
In cystic fibrosis patients, there is a challenge in getting any gene therapy vector intact to the target cells because of the accumulation of extracellular mucus. This experiment demonstrates that the complexes of the invention can meet that challenge. A transwell mucus RTN penetration assay was carried out in 24-well plates with transwells (6.5 mm, 3.0 um pore polyester membrane inserts, Corning, UK) and kept in a 37° C. incubator. Tris-buffer was added to each well (receiver solution) and the transwell was placed on top of the buffer. 1 μl of CF or non-CF (normal) mucus (Epithelix Sarl, Geneva, Switzerland) was added on top and spread across each transwell. The plate was incubated for 30 minutes to equilibrate the mucus. 140 ng/μl siRNA RTNs were prepared and 3 ul were added to each transwell. After 5, 10, 15, 30, 45, 60 and 120 minutes, 100 μl of the tris buffer was collected and pipetted into a 96-well plate. siRNA was measured in a FLUOstar OPTIMA Microplate reader. Cumulative RTN penetration through the mucus was calculated and plotted in
3
Investigating ASC Knockdown Effects on Cell Interactions
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Mouse BV2 cells and human SH-SY5Y cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Beijing, China) and cultured in DMEM supplemented with 10% FBS in 5% CO2 at 37 °C. ASC knockdown in BV2 cells was performed using si-PYCARD/ASC and an RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol. At 48 h post-transfection, cells lysates were collected for IB to verify the transfection efficiencies of knockdown. To determine the effects of ASC knockdown in BV2 cells on SH-SY5Y cells, SH-SY5Y cells in the bottom and BV2 cells on top were separated in a trans-well culture with 0.4 μm pore polyester membrane inserts (Corning cat# 3470) in co-culture conditions. BV2 cells with or without ASC gene knockdown were stimulated with PFFs after which cell lysates and supernatants were collected after 16 h of co-culture.
4
Differentiation of Primary Human Nasal Epithelial Cells
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Primary human nasal epithelial cells (HNE), from PromoCell, were cultured in suspension in PneumaCult Plus Medium according to manufacturer instructions (StemCell Technologies, Cambridge, MA, USA). To generate air-liquid interface cultures, HNE cells were plated on 0.4 μm pore polyester membrane inserts with a 0.4-micron pore size (Corning, Tewksbury, MA, USA) at 3.3 x 104 cells in 0.2 mL PneumaCult-Ex Plus Medium and inserted into 24 well culture plates. Cells were maintained for the first 2~4 days in PneumaCult Plus Medium, until confluence is reached. Medium were gently aspirated from both the basal and apical chambers, then 0.5 ml PneumaCult-ALI Maintenance Medium was added to the basal chamber only. Cells were incubated at 37°C and full medium change in the basal chamber with PneumaCult-ALI Maintenance Medium was performed every 2 days. HNE cells were maintained at air-liquid interface for 28 days for differentiation.
5
Transwell Migration Assay for MDA-MB-231 Cells
6
Evaluating Angiogenic Potential of Cardiac Cells
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HUVECs cocultured with either media control alone, Scr CDCs or nSMase2 KD CDCs on transwell inserts (0.4μm Pore Polyester Membrane Insert, Corning) for 24 hours were trypsinized and seeded at a density of 5 × 103 cells per well in a 96 well tissue culture plate coated with 50ul of growth factor depleted Matrigel (BD Biosciences). Cells were maintained in EBM2 supplemented with an additional 10% of exosome free FBS and 50ng/ml VEGF at 37°C and 5% CO2. After incubating for 4h, tube formation by endothelial cells was photographed (Nikon inverted microscope, 4x objective) and quantified by measuring the length of tubes with NIH Image and counting the branch points in 10 random fields. Experiments were performed in triplicate using CDCs at P4. Significant differences in vessel length and number of branch points with each treatment was determined using a one-way ANOVA with a Tukey’s post-hoc test, p<0.05.
7
Transwell Invasion Assay for CD63 Modulation
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The Transwell invasion assay was performed to examine the invasion ability of CD63-silenced and CD63-overexpressing TCA8113 cells, using a 6.5-mm Transwell with an 8.0-µm Pore Polyester Membrane Insert (Corning Incorporated) coated with 10 µl Matrigel (50 µl/cm2; Corning Incorporated). A total of 1×105 cells were plated into the upper chamber of the Transwell with 500 µl RPMI 1640 medium without FBS, and 500 µl RPMI 1640 medium with 10% FBS was added into the lower chamber. The cells were cultured for 24 h in 5% CO2 at 37°C.
The non-invading cells in the upper side of the filter were then gently removed with a soft cotton swab, and the cells that had invaded to the lower side of the filter were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 1% crystal violet at 37°C for 15 min (Sigma-Aldrich; Merck KGaA). The number of cells in three randomly selected fields was counted with an Image Analysis System (version 3.3.0; Leica Microsystems GmbH), and these numbers are expressed as the average number of migrating cells.
The non-invading cells in the upper side of the filter were then gently removed with a soft cotton swab, and the cells that had invaded to the lower side of the filter were fixed with 4% paraformaldehyde at room temperature for 30 min and stained with 1% crystal violet at 37°C for 15 min (Sigma-Aldrich; Merck KGaA). The number of cells in three randomly selected fields was counted with an Image Analysis System (version 3.3.0; Leica Microsystems GmbH), and these numbers are expressed as the average number of migrating cells.
8
Dioscin Modulates LPS-Induced Co-Culture
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This co-culture model was conducted as previously described 30 (link). Briefly, in our experiment, we used 24 mm Transwell® with a 0.4 μm Pore Polyester Membrane Insert from Corning Company. LPS-primed RAW264.7 cells seeded in the lower well were first treated with CS (50 μg/cm2) for 24 h together with different concentrations of dioscin (300, 150, 75 ng/mL), and then NIH 3T3 cells which had attached on the top of the insert for 24 h were co-cultured with RAW264.7 cells in the Transwell 6-well plate system for another 24 h. After that, NIH-3T3 cells were collected to detect the mRNA levels of collagen I and α-SMA.
9
MDA-MB-231 Cell Migration Assay
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MDA-MB-231 cells were maintained in DMEM media without FBS for 4 h. Approximately 5 × 104 cells of each condition were plated in 100 µL DMEM media without FBS in 6.5 mm Transwell chambers with 8.0 mm Pore Polyester Membrane Insert (Corning Inc., Corning, NY, USA), and transferred to a 24-well dish containing 500 µL fresh media (0.5% FBS containing DMEM) with/without vorinostat. After 24 h, cells were fixed with 4% paraformaldehyde for 30 min, then 0.5% crystal violet (Sigma-Aldrich) was added for 1 h, and washed with PBS. Using a microscope (Nikon, Eclipse TS100), photos of membrane insert were captured at 10× test. ImageJ software was used to quantify migration.
10
Transwell Permeability Assay for Endothelial Cells
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HAOECs were seeded on top of a polyester membrane transwell insert on 12-well-plates (12 mm Transwell® with 0.4-μm Pore Polyester Membrane Insert, Corning, #3460/TC-Inserts, 0.4 mM Pore, Sarstedt, 83.3931.041 for knockdown experiments) at a density of 0,8 × 105 cells/chamber and cultured for 72 h. After discarding the medium, the cells in the upper chamber were incubated with different concentrations of BUT as indicated. Subsequently, the medium in the apical chamber was replaced by endothelial cell basal medium containing 0.05 mg/ml of 10 kDa dextran (Invitrogen, D22910) followed by incubation for 30 min at 37°C in a CO2 incubator, after which the fluorescence (F) intensity in the upper and lower chambers was determined with a 96-well plate reader (Synergy HTX Multifunction Detector, BioTek, United States: excitation 485/20 and emission 528/20). All independent experiments were performed in triplicate. Permeability was presented as Fbasal/Fapical (Fb/Fa). After VEC was knocked down by specific siRNA, permeability was measured as described previously and experiments were performed in duplicate.
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