Migration assays were performed using either scrapping line method or 6.5 mm Transwell® with 8.0 μm Pore Polycarbonate Membrane Insert (Corning, New York, USA) according to the manufacturer’s instructions. At 24 h post-transfection with miR-24 mimics, inhibitors and controls, SGC-7901 cells were incubated in for 24 h, and then 1 × 105 cells in 200 μL serum-free medium were added to the upper chamber. A volume of 500 μL of 10% FBS-containing medium was then added to the lower chamber as a chemo-attractant. Cells were incubated for another 24 h at 37°C, and then non-migrating cells on the upper surface of the membrane were gently scraped off with cotton swabs. Cells that migrated to the bottom of the membrane were stained with the cell stain provided in the assay kit for 20 min and visualized under a microscope. To minimize the bias, at least three randomly selected fields with 200× magnification were counted, and the average number was taken.
Transwell with 8.0 μm pore polycarbonate membrane insert
Manufactured by Corning
Sourced in United States
The Transwell with 8.0-μm pore polycarbonate membrane inserts is a cell culture product used for various applications. The inserts feature a polycarbonate membrane with 8.0-μm pore size, enabling cell migration and invasion studies.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (6 mentions)
Fsx100 microscope, by Olympus (3 mentions)
Biocoat matrigel invasion chamber, by Corning (2 mentions)
Matrigel, by BD (1 mentions)
Quercetin, by Merck Group (1 mentions)
Anti α tubulin antibody, by Merck Group (1 mentions)
Radio immunoprecipitation assay ripa lysis buffer, by Beyotime (1 mentions)
8.0 μm pet membrane, by Corning (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Growth factor reduced matrigel, by Corning (1 mentions)
Paraformaldehyde (pfa), by Biosharp (1 mentions)
Giemsa, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Matrigel, by Thermo Fisher Scientific (1 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (1 mentions)
Matrigel invasion chamber, by BD (1 mentions)
Te200 microscope, by Nikon (1 mentions)
Crystal violet, by Beyotime (1 mentions)
Fibronectin, by Merck Group (1 mentions)
23 protocols using transwell with 8.0 μm pore polycarbonate membrane insert
1
Evaluating Cell Migration via Transwell Assay
2
Measuring Cell Migration and Invasion
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Cell migration and invasion were also measured using sterile Transwell with 8.0 μm pore polycarbonate membrane insert (Corning Incorporated, Corning, NY, USA). Similarly, after maintaining PC3 cells under serum starvation conditions for 24 h, the cells (2 × 104 cells/well) were digested and loaded onto the top of a 24-well migration chamber in 100 μl serum-free DMEM/F12 medium containing different low concentrations of dioscin (0.35, 0.7 and 1.4 μM), and 0.75 ml medium containing 10% FBS was added to the lower chamber for 24 h culturing. Eventually, the cells that had migrated into the lower surface of the filter were fixed with 10% formaldehyde and stained with hematoxylin after 24 h incubation.
3
Quercetin Inhibits Cell Migration
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Quercetin was purchased from Sigma-Aldrich Co. (St Louis, MO, USA), and Matrigel was obtained from BD Biosciences (San Jose, CA, USA). Transwell with 8.0 μm pore polycarbonate membrane insert was obtained from Corning Incorporated (Corning, NY, USA). Anti-VEGFA (Cat No sc-7269) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Anti-MMP2 (Cat No 10373-2-AP) and anti-MMP9 antibodies (Cat No 10375-2-AP) were purchased from Proteintech, Chicago, China. Anti-α-tubulin antibody was obtained from Sigma-Aldrich Co. Radioim-munoprecipitation assay (RIPA) lysis buffer was purchased from Beyotime (Nantong, China). This study was approved by the Institutional Review Board of Ethics Committee of Renmin Hospital, Hubei University of Medicine.
4
Cell Migration and Wound Healing Assay
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One day post transfection of JHH6 cells, culture medium was replaced with fresh medium without FBS, for cell starvation. One day later, cells were washed, trypsinized, and counted. Then, 105 cells were resuspended in 150 μL of fresh medium without FBS and placed into transwells (6.5-mm Transwell with 8.0-μm pore polycarbonate membrane insert; 3422 Corning). As a migration stimulus, 600 μL of fresh medium with 20% FBS was used. Non-transfected starved cells were used as an additional control and were assayed with medium with 20% FBS (positive control) or no FBS (negative control) as stimuli. JHH6 cells were allowed to migrate for 24 h. Finally, culture medium was aspirated, membranes were carefully washed three times with 300 μL of PBS, and cells were fixed with 150 μL of 4% PFA for 20 min and stained for 20 min with 0.1% crystal violet diluted in 20% methanol. After staining, membranes were washed again three times with 300 μL of PBS and pictures of each membrane were taken in the bright field of the microscope. The stained area was quantified with FIJI image software analysis.
For the wound healing assay, a wound was made to cells with 80%–90% confluency employing a 200-μL pipette tip. Then, pictures were taken under the bright field of the microscope, right after the wound and 16 h later. Images were analyzed with ImageJ software to quantify the wound area.
For the wound healing assay, a wound was made to cells with 80%–90% confluency employing a 200-μL pipette tip. Then, pictures were taken under the bright field of the microscope, right after the wound and 16 h later. Images were analyzed with ImageJ software to quantify the wound area.
5
Cell Migration and Invasion Assays
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Cell migration assays were performed using 6.5 mm Transwell with 8.0 μm pore polycarbonate membrane insert (Corning) as previously described [9 (link)] with some modifications. In brief, cells (1.0 × 105) in 200 μl serum-free RPMI-1640 medium were loaded into the top chambers, and 600 μl RPMI-1640 media containing 10% FBS was added into the lower chamber as a chemoattractant. After incubation for forty-eight hours at 37°C in 5% CO2, the non-migratory cells on the upper surface were removed by a cotton swab and the cells that had migrated to the other side of the membrane were fixed with 4% paraformaldehyde for 20 minutes and stained with Wright-Giemsa. Values for migration were obtained by counting cells in 10 fields (2 × (centre + 4 quadrants)) per membrane (× 20 objective) and averaged for three wells. For cell invasion assays, cells (1.0 × 105) were suspended in 200 μl serum-free RPMI-1640 and seeded on the matrigel-coated membrane in each insert of biocoat matrigel invasion chambers with 8.0 μm PET membrane (Corning). Forty-eight hours later, the cells that had invaded the matrigel and moved to the other side of the membrane were fixed, stained and counted as in the migration assay. The experiment was repeated three times independently.
6
Transwell Migration Assay for Single Cells and Clusters
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Migration of single cells and clusters was evaluated by using 6.5 mm Transwell®® with 8.0 μm Pore Polycarbonate Membrane Insert (Corning, Madrid, Spain). Cells were cultured overnight under suspension and serum starvation conditions. For invasion assays, each Transwell was coated with Growth Factor Reduced Matrigel (Corning) and incubated for 3 h to allow Matrigel polymerization. Cells were cultured overnight under suspension and serum starvation conditions. The day after, 5 × 104 cells/transwell were seeded as both single and cluster suspension. Assays were incubated with a 10% gradient of FBS (37 °C, 5% CO2), during 8 h for MDA-MB-231 cell line, or 24 h in the case of MCF7 cells. After the incubation, transwell membranes were scraped in order to remove the non-migrated cells. Migrated cells were fixed with 3% w/v PFA + 1% v/v glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 15 min. Migrated eGFP-expressing cells were counted using a microscope Leica DMi8 (Leica Microsystems, L’Hospitalet de Llobregat, Spain) and the free software ImageJ (NIH Image, Bethesda, MD, USA).
7
Transwell Migration and Invasion Assay
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Transwell assays were used to evaluate Huh-7 and Hep3B cells migration and invasion. For cell migration, 100 μL DMEM without FBS, containing 20,000 cells, was incorporated to the upper chambers (6.5 mm Transwell with 8.0 μm pore polycarbonate membrane insert, Corning, USA, Cat#CLS3422) of the Transwell device. Subsequently, 600-μL DMEM with 10% FBS was incorporated to the lower chamber. For the invasion assay, 100 μL DMEM without FBS was incorporated to the upper chambers to rehydrate Matrigel (Cat #356234; Corning, USA) for 2 h at 37 °C. After removing the supernatant, 40,000 cells containing DMEM without FBS were seeded into the Matrigel-pretreated upper chambers, and 600 μL DMEM with 10% FBS was incorporated to the lower chamber. Following 24-h of incubation, migrating or invading cells were fixed in 4% paraformaldehyde (Biosharp, China, Cat#BL539A) for 20 min and stained with Giemsa (Beyotime, China, Cat#C0121-100 ml). An inverted electron microscope (OLYMPUS IX71, OLYMPUS, Japan) was used to obtain the final images. The number of cells was enumerated.
8
Transwell Assay for Cell Invasion
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Transwell with 8.0 μm pore polycarbonate membrane insert and 1 × 105 pores per cm2 (Corning, Inc., Corning, NY) was used. RCC cells (1 × 104 cells/well) were harvested and seeded with serum-free DMEM medium into the upper chamber, and the lower chamber contained DMEM medium with 10% FBS. After 6 h incubation at 37 °C, the invaded cells were fixed with paraformaldehyde and stained with crystal violet. Cell numbers were counted in five randomly chosen microscopic fields.
9
Tumor Cell Invasion Assay Protocol
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A 6.5 mm transwell with 8.0 μm pore polycarbonate membrane insert (Corning, USA) was utilized in invasion assay. Firstly, 100 μL diluted growth factor reduced matrigel basement matrix (Corning, USA) was added into transwell inserts for one hour at 37 °C. A 100 µL 3 × 105/mL tumor cell suspension was seeded into upper chamber coated matrigel in serum-free DMEM medium, and 600 µL DMEM containing 10% FBS medium was added into lower chamber. After that, cells' suspension in upper chamber was removed and transwell membrane was gently wiped with a moistened cotton swab to remove the matrigel and cells. Next, 4% paraformaldehyde was used to fix the cells in transwell membrane for 30 minutes, and 1% crystal violet was used to stain membrane for 5 minutes. After washing three times, transwell membrane was dried and photographed under a microscope to count cell number.
10
Quantifying Cell Invasion and Migration
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Cells were infected with shControl or shMerTK for > 72 h or treated with vehicle or indicated concentrations of UNC2250 for 2 h in FBS-free DMEM. For invasion assays, cells were seeded into Corning BioCoat Matrigel invasion chambers with an 8.0-μm polyethylene terephthalate membrane (Costar; Corning Incorporated, Corning, NY, USA) in 24-well plates; for migration assays, cells were seeded into Transwell with 8.0 μm pore polycarbonate membrane insert (Costar; Corning Incorporated, Corning, NY, USA). Cells in the upper chamber were cultured in FBS-free DMEM, while 30% FBS was added to the lower chamber. After 24 h, cells invading or migrating into the lower chamber were harvested and resuspended in 100 μl DMEM. Afterwards, cells were plated in 96-well black base microplates, and viable cells were measured as in the cell proliferation assays. Invasive abilities or migration abilities were determined by the number of viable cells invading or migrating into the lower chamber.
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