Both assays used a transwell membrane (8 μm pore size, 6.5 mm diameter; Corning Costar). A total of 2.5 × 104 cells were plated in the top chambers for the transwell migration assay. The top chambers were filled with serum-free medium, and the bottom chambers were filled with migration-inducing medium (with 10% FBS). The filters were fixed with 4% paraformaldehyde after 24 h. The cells on the upper side of the membrane were scraped with a cotton swab, and the cells on the bottom side of the membrane were stained with 4′,6-diamidino-2-phenylindole (DAPI). The membranes were washed with PBS and photographed after drying. The top chambers were coated with Matrigel before 2.5 × 104 cells were plated for the Matrigel invasion assay. Images were taken after 72 h.
Transwell membrane
Manufactured by Corning
Sourced in United States, United Kingdom, Japan, Germany
Transwell membranes are a type of lab equipment used for cell culture experiments. They consist of a porous membrane that allows the passage of media and molecules between the upper and lower chambers, enabling the study of cell migration, permeability, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (18 mentions)
Matrigel, by Corning (12 mentions)
Inverted microscope, by Olympus (7 mentions)
Crystal violet, by Merck Group (5 mentions)
Type 1 collagen, by BD (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Growth factor reduced matrigel, by BD (3 mentions)
Dm5000b, by Leica (3 mentions)
Rat tail collagen type 1, by Thermo Fisher Scientific (3 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Spectrafluor plus, by Tecan (2 mentions)
Fibronectin, by Roche (2 mentions)
Image pro plus, by Media Cybernetics (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Pneumacult ali medium, by STEMCELL (2 mentions)
Pneumacult ali, by STEMCELL (2 mentions)
Recombinant epidermal growth factor and bovine pituitary extract, by Thermo Fisher Scientific (2 mentions)
Snapwell membranes, by Corning (2 mentions)
159 protocols using transwell membrane
1
Transwell Migration and Invasion Assays
2
Wound Healing and Transwell Migration Assays
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For wound-healing migration assay, confluent monolayers of cells were wounded with a p20 pipette tip. The images were taken after PBS washes (time 0 h). The cells were cultured in serum-free medium. After two days, the images were taken again (time 48 h). Three separate fields were photographed for each plate. For Transwell migration assay, Transwell membrane (8 μm pore size, 6.5 mm diameter; Corning Costar) was used for assay. Briefly, 2.5 × 104 cells were plated in the top chambers. The top chambers were filled with serum-free medium and the bottom chambers were filled with migration-inducing medium (with 10% FBS). The filters were fixed with 4% paraformaldehyde after 24 h. The cells on the upper side of the membrane were scraped with a cotton swab and the cells on the bottom side of the membrane were stained with crystal violet. The membranes were washed with PBS and images were taken after drying out.
3
Brain Endothelial Cell Monolayer Formation
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Primary brain endothelial cells BECs (5 × 104 cells) were plated on the upper side of a collagen and fibronectin-coated polyester Transwell membrane (Costar, pore size 3 μm; growth area 1.12 cm2) in endothelial basal medium. Cells were then incubated at 37°C in a 5% CO2 atmosphere. Under these experimental conditions, brain endothelial cells formed a confluent monolayer within 12 days.
4
Boyden Chamber Assay for OEC Migration
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The modified Boyden chamber assay 22 was used. Briefly, transwell inserts (Costar Transwell membrane, 6.5 mm diameter, 12.0 μm pore size) were placed in 24‐well dishes. Rabbit PB OECs were plated on the upper wells (5 × 104 cells/well) to culture for 24 hours in EBM‐2 containing FBS (5%) and stromal cell‐derived factor‐1α (SDF‐1α, 100 ng/mL; Sigma‐Aldrich). Adding bilobalide and L‐NAME into the media of related groups refers to the above description. Cells that migrated to the underside of the membrane were Hoechst‐stained for counting.
5
Cell Migration and Invasion Assays
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Transwell membrane (8 μm pore size, 6.5 mm diameter) (Corning Costar, Corning, NY, USA) was used for both assays. For transwell migration assay, 2.5 × 104 cells were plated in the top chambers. Serum-free medium was added into the top chambers and migration-inducing medium (with 10% FBS) was added into the bottom chambers. After 24 h, the filters were fixed with 4% paraformaldehyde. Cotton swabs were used to scrape the cells on the upper side of the membrane and crystal violet was used to stain the cells on the bottom side of the membrane. The membranes were washed with PBS and photographed after dry out. For Matrigel invasion assay, the top chambers were coated with Matrigel before 5 × 104 cells were plated. Images were taken after 24 h.
6
Cytokine-Induced Migration Assay
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The migration assay was performed using a trans-well membrane (8 μm pore size; Corning Costar Corp., New York, NY) in 24-well plates. The SPCs were seeded onto the upper chamber, and media containing various concentrations of recombinant human cytokines (rhCXCL6, rhIL8 and rhCCL5) or ECFCs were placed in the lower chamber. The plates were incubated for 24 hr, and the migrated cells were fixed and stained. Cells that had migrated to the lower surfaces of the membranes were quantified under a microscope. The same studies were conducted for CXCL6 and IL8. All assays were conducted in triplicate.
7
Migratory and Invasive Abilities of Treated Cells
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The migratory ability of DMSO- and coriloxin-treated cells was assessed using the protocol of the wound healing assay, as described elsewhere [34 (link)]. The cells that had migrated into the zone previously empty of cells at indicated times were counted through microscopic observation. Through the transwell membrane assay, performed using a transwell membrane (pore size 8 μm; Corning Costar, Cambridge, MA, USA) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) [34 (link)], the cells’ invasion ability was examined. After 18 h of incubation, cells adhering to the polycarbonate filter’s lower surface were counted through light microscopy (magnification 200×) and subsequently photographed. We conducted the experiments in triplicate.
8
HUVEC Permeability Assay Using Rhodamine-Dextran
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For in vitro permeability assay, the pre-treated HUVECs were plated and allowed to reach confluence on the top of the transwell membrane (diameter: 0.4 μm; Corning-Costar, NY, USA). Subsequently, rhodamine-dextran (average MW ~70,000; Sigma, USA) was added in the upper chamber at 20 mg/ml for 30 min. 40 μl medium of the lower chamber was absorbed and measured under 544 nm excitation and 590 nm emission.
9
Triptolide Modulates VSMC Migration
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Vascular smooth muscle cell migration was determined using Transwell chambers, with a Transwell membrane containing 8-μm pores (Costar; Corning) and coated with 10 μg/mL fibronectin, inserted into a 24-well plate. The lower chamber was filled with 600 μL of DMEM with 10% FBS, and VSMCs (1 × 105 cells/well) in serum-free DMEM were placed in the upper chamber, followed by incubation with PBS or various concentrations of triptolide (0, 5, and 10 ng/mL) for 24 h. Infiltrated cells were fixed in 4% paraformaldehyde (Bioss, C01-06002) and stained with 0.1% crystal violet (Bioss, D10162). Migrated cells were photographed under an inverted microscope (LEICA DMI 4000B, Germany), and five random high-power fields (200× magnification) were selected for quantification of cell number using the IPP 6.0 imaging software (Media Cybernetics). VSMC-migration ability was expressed as the ratio of the number of migrated cells to that of control cells.
10
Transwell Assay for Cell Invasion and Migration
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A Transwell membrane (Costar; Corning Incorporated, Corning, NY, USA) was used for cell invasion and migration assays, according to the manufacturer's protocol. Following treatment with various concentrations of negative group (wild-type MGC-803 cells), COE group (20, 40 and 80 µg/ml) and 5-fluorouracil (5-FU) positive group (32 µg/ml) for 24 h, cells were seeded in the upper part of a Matrigel-coated invasion chamber in a serum-free medium. Medium containing 20% FBS was applied to the lower chamber. After 24 h, the cells remaining in Matrigel were removed by scraping, while the cells that invaded through Matrigel were fixed and stained by using 0.5% crystal violet (Beyotime Institute of Biotechnology) in methanol for 30 min. Images were captured under a fluorescence microscope at magnification, ×400 (Nikon Corporation, Tokyo, Japan) and invading cells were quantified by manually counting 5 fields of view. Migration assays followed in the same procedure, but with no Matrigel coating on the polycarbonate membrane. Each experiment was repeated three times.
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