The cell invasion assay was performed using 24-well transwell insert chambers with a polycarbonate filter with a pore size of 8.0 μm (Costar, Corning, NY). Two × 104 cells in serumfree medium were seeded on top of a Matrigel-coated filter (BD Biosciences, Franklin Lakes, NJ) in each insert chamber. Then, the insert chambers were placed into wells on top of culture medium containing 10% fetal bovine serum as a chemoattractant. The invasive ability of cells was determined by the number of cells translocated to the lower side of filters.
Transwell chambers 24 well insert
Manufactured by Corning
Sourced in United States
Transwell chambers (24-well insert) are a type of laboratory equipment used for cell culture and migration studies. These chambers consist of a 24-well insert with a semi-permeable membrane that separates the upper and lower compartments, allowing for the study of cell behavior and interactions between different cell types or conditions.
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9 protocols using transwell chambers 24 well insert
1
Cell Invasion Assay Protocol
2
Cell Invasion and Migration Assay
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The cell invasion assay was performed using 24-well transwell insert chambers with a polycarbonate filter with a pore size of 8.0 µm (Costar, Corning, NY). A total of 2×104 cells in serum-free medium were seeded on top of a Matrigel-coated filter (BD Biosciences, Franklin Lakes, NJ) in each insert chamber. Then, the insert chambers were placed into wells on top of culture medium containing 10% horse serum as a chemoattractant. The migration assay was performed using 24-well transwell insert chambers with a polycarbonate filter without Matrigel. The invasive or migratory ability of cells was determined by the number of cells translocated to the lower side of filters [35] (link), [36] (link).
3
Transwell Assay for Cell Migration and Invasion
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Cell migratory and invasive abilities were measured using the 24-well Transwell insert chambers (Corning Inc. New York, NY, USA) with the filtration membrane pore size of 8 µm. Prior to cell invasion assays, the membranes were coated with Matrigel (BD Bioscience, San Diego, CA, USA). Transfected cells in serum-free medium were seeded in the upper chambers and medium supplemented with 10% FBS was added to the lower chambers. After 24 h of incubation, non-migrated or non-invaded cells were removed using a cotton swab and migratory or invasive cells were fixed, stained, imaged, and counted under a light microscope.
4
Cell Migration Assay of NSCLC
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NSCLC cells (1 × 105) were plated into the upper chamber of the 24-well transwell insert chambers (Corning, NY, USA). Chambers were precoated with 30 ul Matrigel (BD Bioscience, CA, USA), which was diluted with serum-free medium (ratio of 1:8) and then incubated at 37 ℃ for 1 h. After incubation for 48 h, cells that migrated to the lower chamber were fixed with 4% paraformaldehyde (Biosharp, China) for 20 min and stained with 0.1% crystal violet (Solarbio Lifer Science, China) for 10 min. Five randomly chosen visual fields were counted and imaged with the microscope.
5
Cell Proliferation and Migration Assay
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For the evaluation of cell proliferation, we seeded 3,000 cells/well in triplicates in 96-well pre-coated culture plates. Twenty-four, 48, 72, and 96 h after transfection, 10 µL of Cell Counting Kit-8 reagent (CCK-8, Dojindo, Kumamoto, Japan) was added to the medium and incubated for 3 h at 37 °C. The absorbance at 450 nm was measured using a Multiskan GO microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). This experiment was repeated 3 times.
Cell migration was evaluated using Transwell chambers (24-well insert; Corning, Inc., Corning, NY, USA). A total of 50,000 Jurkat, Jurkat-shLuc, and Jurkat-shRNA-B7H6 cells suspended in RPMI-1640 containing 1% FBS and seeded into the upper chamber of the insert 48 h after transfection. RPMI-1640 containing 15% FBS was added to the lower chamber to serve as a chemoattractant. Cells were allowed to migrate for 24 h. The cells on the upper surface of the membrane were collected using a cotton bud along with the migrating cells in the lower chamber. These cells were subjected to light microscopy and photographed (COIC, Chongqing, China). In some experiments, we added Matrigel (BD Bioscience, Corning, NY, USA) to evaluate cell invasion. These experiments were repeated three times.
Cell migration was evaluated using Transwell chambers (24-well insert; Corning, Inc., Corning, NY, USA). A total of 50,000 Jurkat, Jurkat-shLuc, and Jurkat-shRNA-B7H6 cells suspended in RPMI-1640 containing 1% FBS and seeded into the upper chamber of the insert 48 h after transfection. RPMI-1640 containing 15% FBS was added to the lower chamber to serve as a chemoattractant. Cells were allowed to migrate for 24 h. The cells on the upper surface of the membrane were collected using a cotton bud along with the migrating cells in the lower chamber. These cells were subjected to light microscopy and photographed (COIC, Chongqing, China). In some experiments, we added Matrigel (BD Bioscience, Corning, NY, USA) to evaluate cell invasion. These experiments were repeated three times.
6
Transwell Migration Assay Protocol
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Transwell chambers (24-well insert; Corning, NY, USA) were used to analyze cell migration in accordance with the manufacturer's instructions. Cells were seeded in the upper well of the chamber in DMEM or RPMI-1640 medium without serum, while the lower chamber well contained DMEM or RPMI-1640 medium supplemented with 10% FBS to stimulate cell migration. After incubation for 12 h, cells on the upper membrane surface were removed while the bottom cells were fixed with 3% paraformaldehyde, stained with 0.1% crystal violet, and 5-6 random fields were collected for quantification for each well. The cells were finally extracted with 33% acetic acid and detected quantitatively using a standard microplate reader (at 570 nm). Three independent experiments were performed for each experimental group.
7
Quantifying Cell Invasion in Transwells
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Cell invasion was quantified using SACC cell lines in Transwell chambers (24-well insert; 8 µm pore size; Corning Incorporated, USA). BD Matrigel™ (BD Biosciences Inc., USA) was thawed at 4 °C and then diluted with cold DMEM at a ratio of 1:5. Twenty-five microliters of the diluted Matrigel matrix was added to the Transwell membrane inserts. When the plates were incubated for 6 h at 37 °C, 2 × 105 cells in 200 µl of serum-free DMEM were seeded onto the Matrigel-coated upper chamber, and the lower chamber was filled with 500 µl of DMEM with 10% serum. After 24 h invading, the cells on the lower side of the chamber were fixed with methanol for 10 min and stained with 0.5% crystal violet for 15 min. Five random fields on the lower side of the chamber were selected for quantification under an inverted microscope (200 × ).
8
Transwell-based Cell Migration Assay
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Transwell chambers (24-well insert; Corning, Inc.) were used to analyze cell migration. At 48 h post-transfection, a total of 2×104 C666-1 cells in serum-free RPMI-1640 medium were seeded into the upper chamber of the insert. RPMI-1640 medium containing 10% FBS was added to the lower chamber to serve as a chemo-attractant. Cells were allowed to migrate for 24 h. The cells on the upper membrane surface were removed using a cotton bud and the cells on the lower membrane surface were fixed with 4% formaldehyde. The cells were subsequently stained with 0.1% crystal violet (Amresco, LLC) and the migrated cells were counted in three random-selected fields. The result of migrated cells was observed and photographed under light microscope (Olympus, Japan).
9
Transwell Cell Migration Assay
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Transwell chambers (24-well insert; Corning, NY, USA) were used to for cell migration assays. After transfection, cells were suspended in medium without serum and added to the upper chambers. After migration for 12 h, cells on the upper membrane surface were removed. The lower membrane surface was fixed with 4% formaldehyde, stained with Hoechst and counted under a fluorescence microscope. And 5-6 random fields were collected for quantification.
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