Tumor cell invasion and migration capacities were assessed using Transwell inserts with 8 μm pores (Corning, NY). To assess invasion capacity, after transfection for 24 hours, cells (3.0 × 105) cultured in serum‐free medium were added to the apical chamber that was pre‐coated with Matrigel matrix (BD, Franklin Lakes, NJ). FBS (10%, 500 μl) was dispensed in the matching basolateral chamber. After incubation for 48 hours, non‐invading cells that remained on the upper chamber surface of the transwell membrane were removed using a cotton swab. Invading cells that had traversed the membrane were fixed with methanol and stained with 0.1% crystal violet. Images were obtained and the cells were enumerated. The migration assay was performed the same way, except that 2 × 105 cells were added into the chambers without a pre‐coated matrix gel. Six fields at 100× magnification were randomly selected and the cells were counted. Each experiment was conducted three times.
Transwell inserts with 8 μm pores
Manufactured by Corning
Sourced in United States
Transwell inserts with 8-μm pores are a laboratory equipment used for cell culture and migration studies. The inserts feature a semi-permeable membrane with pores of 8 micrometers in diameter, which allows for selective passage of cells and molecules between the upper and lower chambers of the Transwell system.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel matrix, by BD (4 mentions)
Crystal violet, by Merck Group (1 mentions)
24 well cell culture plate, by Corning (1 mentions)
Sflt 1, by R&D Systems (1 mentions)
Celtrex reduced growth factor bm extract pathclear, by Bio-Techne (1 mentions)
Accutase, by Innovative Cell Technologies (1 mentions)
C7661, by Merck Group (1 mentions)
Evom2, by World Precision Instruments (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Matrigel, by BD (1 mentions)
Matrigel, by Corning (1 mentions)
19 protocols using transwell inserts with 8 μm pores
1
Tumor Cell Invasion and Migration Assay
2
Evaluating Invasive Potential of Cells
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The invasive and migratory potential of cells was evaluated using Transwell inserts with 8-μm pores (Corning, Corning, NY, USA). For the invasion assay, 3.0 × 105 cells in serum-free medium precoated with Matrigel matrix (Becton, Dickinson and Company, East Rutherford, NJ, USA) were added to each upper insert 24 h after transfection, and 500 μl of 10% FBS medium was added to the matched lower chamber. After 48 h of incubation, cells that did not invade were removed from the upper surface of the Transwell membrane with a cotton swab, and cells on the lower membrane surface were fixed in methanol, stained with 0.1% crystal violet, and photographed. The staining was then dissolved in 5% acetic acid, and the OD at 490 nm value in each well was determined by a microplate reader.
3
Quantifying Cell Growth, Colony Formation, and Invasion
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Cell growth was determined using the Cell Counting Kit-8 method (MCE, Monmouth Junction, US) according to the manufacturer's protocol. For colony formation assays, cells were trypsinized and seeded in 6-well plates at a density of 3 × 102 cells per well and cultured at 37˚C for 10 days. The colonies were stained with 0.1% crystal violet solution containing 80% methanol for 5 min at room temperature. Colonies with > 50 cells/colony were counted at 40× magnification using a light microscope. Cell invasion was measured using Transwell inserts with 8 μm pores (Corning, US). Cell density was adjusted to 106/mL in serum-free RPMI-1640 medium and 200 μL of cell suspension was added to each upper insert pre-coated with Matrigel matrix (BD, US); 500 μL RPMI-1640 medium containing 10% FBS was added into a matched lower chamber. After a 48 h incubation, the non-invading cells were removed from the upper surface of the transwell membrane with a cotton swab and the invading cells on the lower membrane surface were fixed in methanol, stained with 0.1% crystal violet, photographed, and counted. Six random fields for each insert were observed at 100× magnification.
4
Macrophage Chemotaxis Modulated by ASC Secretome
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The influence of conditioned medium derived from monolayer ASCs and ASC sheets on the chemotaxis of mouse macrophage J774A1 cells (Bioresource Collection and Research Center, Hsinchu, Taiwan) was determined using transwell inserts with 8-μm pores (Corning). J774A1 cells which had been stimulated by lipopolysaccharide (LPS; 0.1 μg/mL) for 4 h were seeded in the lower wells at a density of 3 × 105 cells/well. The upper wells were seeded with unstimulated macrophages at a density of 1 × 105 cells/well, and the medium was replaced with the conditioned media of FBS or HPL-cultured monolayer ASCs or ASC sheets. After 24 h of culture in conditioned medium, macrophage migration through the transwell membrane was assessed through crystal violet (Sigma) staining of the lower surface of the membrane. The stained cells in the acquired microscopic images were quantified using ImageJ.
5
Evaluating Invasive Potential of Cells
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The invasive and migratory potential of cells was evaluated using Transwell inserts with 8-μm pores (Corning, Corning NY, USA). For the invasion assay, 24 h after transfection, 3.0 × 105 cells in serum-free medium were added to each upper insert precoated with Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA). Five hundred microliters of 10% FBS medium was added to the matched lower chamber. After 48 h of incubation, cells that did not invade were removed from the upper surface of the Transwell membrane with a cotton swab, and cells on the lower membrane surface were fixed in methanol, stained with 0.1% crystal violet, and photographed. The staining was then dissolved by 5% acetic acid, and the OD 490 nm value in each well was determined by a microplate reader.
6
Transwell Invasion and Migration Assay
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Cell invasion was evaluated using Transwell inserts with 8 μm pores (Corning, Inc., Corning, NY, USA). Briefly, 48 h after transfection, the NCAPG siRNA-treated cells and control groups cells were re-suspended in DMEM medium without serum and growth factor, and then 200 μl cell suspension was seeded in the upper Transwell chambers. Subsequently, 500 μl medium containing 20% FBS was added to the bottom of the chambers. The 24-well culture plate was incubated at 37°C in a humidified atmosphere containing 5% CO2 for 48 h. Cells remaining on the upper surface of the Transwell chambers were mechanically removed using a cotton swab, and then the underside of the Transwell chambers was fixed, washed and stained by hexamethyl pararosaniline. Five random fields on the lower surface of each Transwell chamber were counted. The migration assay procedures differed from the invasion assay, in that they were performed in the Transwell chambers that were not pre-coated with Matrix gel.
7
Quantifying HT1080 Cell Migration
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HT1080 cells were incubated with the media containing 10 ng/ml TNFα for 24 h. A single cell suspension of HT1080 cells was obtained via trypsin-EDTA treatments for 2 min at 37°C. For the migration assay, transwell inserts with 8 μm pores (Corning Inc., USA) were purchased from Invitrogen (USA). Cells (2 × 104) in RPMI were added to the transwell inserts. The bottom of the wells was filled with RPMI containing 10% FBS. Plates were maintained in a humidified incubator with 5% CO2 at 37°C for 24 h. Transwell inserts were washed in PBS, and cells in the inner well that had not migrated were removed with a cotton swab. Membranes were fixed in 4% formalin and stained with hematoxylin, and then cells that had migrated were counted in 5 high power fields under the microscope.
8
Macrophage Migration Assay with DFSC-CM
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Macrophages was seeded at 1 × 106 cells/mL into the upper chamber in a total volume of 250 μL. Transwell inserts with 8 μm pores (Corning, NY, USA) were used. Sh-nc DFSC-CM or sh-postn DFSC-CM was added to RPMI 1640 containing 250 ng/mL P.g-LPS at a 1:1 ratio (500 µL total) to the lower chamber. The group to which only P.g-LPS-containing RPMI 1640 was added was used as the control. Macrophages were allowed to migrate through the transwell insert membrane for 24 h. The inserts were removed, fixed in cold methanol, and stained with methanol containing 1% crystal violet. The inside of the Transwell was swabbed thoroughly with cotton swabs and air dried at room temperature overnight. The number of migrated macrophages was counted by bright-field microscopy at 4× and 10×. Five bright-field images were analyzed per well.
9
In vitro Cell Migration Assay
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In vitro cell migration assays were performed in 24-well plates using transwell inserts with 8 μm pores (Corning, Corning, NY) following the procedures described previously [41 (link)]. Briefly, M0 cells were seeded (5 X 104 cells/insert) onto the upper well of the chamber, and the inserts were placed in 24-well cell culture plates (Corning) containing 500 μL of RPMI-1640 medium with or without 2 μg/ml rhVEGF, and incubated at 37°C in 5% CO2 for 16 hours. In another set of experiments, the cells were incubated in RPMI-1640 medium with concentrated supernatants from decidualized endometrial stromal cells and with or without sFlt1 (1.5 μg/ml; R&D systems), as described above. Concentrated supernatants from nondecidualized stromal cells were used as controls. The cells in the upper chambers were removed carefully with a wet cotton swab. The migrated cells on the bottom surface were stained with Diff-Quick Hema3 stain following the manufacturer’s instructions (Fisher Scientific, Kalamazoo, MI), and six evenly spaced fields of cells were counted in each well using a Leica DMIRES2 microscope.
10
Transwell Invasion Assay for Cells
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Transwell inserts with 8 μm pores (Corning Inc.) were coated with Celtrex Reduced Growth Factor BM extract-PathClear (1:100 in Serum Free media, Trevigen) and allowed to polymerize overnight at 37°C. Cells were gently removed from culture plates using Accutase (Innovative Cell Technologies), and seeded at a density of 20,000 cells per well in serum-free RMPI1640 media. As a chemotactic agent, 10% serum containing medium was seeded on the outside of the transwells. After 24 h, membranes were fixed for 2 min in 100% methanol and stained using Harleco Hemacolor Staining Kit (EMD Chemicals). Invaded cells were counted with ImageJ (43 (link)).
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