Cell migration assays were performed with 24-well transwells (8-μm pore size, Falcon). In total, 1.5 × 105 transfected cells were suspended in serum-free DMEM medium and added to the upper chamber, and 700μL DMEM with 10% FBS was placed in the lower chamber. After 16 h of incubation, cells on the lower surface of membrane were fixed in 4% paraformaldehyde and stained with crystal violet. Cells in six microscopic fields were counted and photographed.
24 well transwell
Manufactured by BD
Sourced in United States
The 24-well Transwell is a laboratory assay system designed for cell-based experiments. It consists of a 24-well cell culture plate with a porous membrane insert that allows for the study of cell migration, invasion, and permeability. The system provides a controlled environment for these types of cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (4 mentions)
Culture insert, by Ibidi (2 mentions)
μ slide angiogenesis, by Ibidi (1 mentions)
Matrigel, by BD (1 mentions)
Spectrophotometer reader, by Thermo Fisher Scientific (1 mentions)
Cck 8 kit, by Yeasen (1 mentions)
Falcon354480, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Axio observer z1 inverted epifluorescence microscope, by Zeiss (1 mentions)
6 well transwell chambers, by BD (1 mentions)
Krn633, by Selleck Chemicals (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ix71 inverted microscope, by Olympus (1 mentions)
Matrix gel, by BD (1 mentions)
Diff quik staining kit, by Sysmex (1 mentions)
14 protocols using 24 well transwell
1
Cell Migration Assay Protocol
2
Cell Invasion and Migration Assays
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The 24-well transwells (8 μm pore size, Falcon, USA) were used for evaluating cell invasion and migration ability. A total of 1 × 105/200 μL cells were cultured in serum-free medium in the upper chambers, with or without Matrigel (Corning, USA), and medium containing 10% FBS was added into the bottom chambers. The non-invasive or non-migratory cells were removed after 12–24 h, penetrated cells were fixed, stained, and counted.
A total of 3 × 104 cells were suspended in 70 μL of RPMI-1640 medium and were seeded in ibidi culture insert (ibidi, Germany). The incubation chambers were placed in a 6-well plate. When cells were at full confluency, the insert was removed to create a gap, and serum-free medium was subsequently added. Image was captured by microscope at 0 h and 24 h, the distance between the gap was measured, the data were collected from three independent experiments and the migration rate of the cells was expressed as relative gap closure by using the ImageJ software.
A total of 3 × 104 cells were suspended in 70 μL of RPMI-1640 medium and were seeded in ibidi culture insert (ibidi, Germany). The incubation chambers were placed in a 6-well plate. When cells were at full confluency, the insert was removed to create a gap, and serum-free medium was subsequently added. Image was captured by microscope at 0 h and 24 h, the distance between the gap was measured, the data were collected from three independent experiments and the migration rate of the cells was expressed as relative gap closure by using the ImageJ software.
3
Exosome-Mediated Angiogenic Potential
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After the pretreatments of HUVECs with various exosomes for 48 h in six‐well plates, the tube formation and migration ability were tested. The 24‐well transwells (8.0 μm, Falcon) were used for assessing migration ability of HUVECs. Pretreated HUVECs were harvested and 2 × 104 cells resuspended in 200 μl ECM without FBS were plated in the upper chamber of a transwell unit, while the lower chamber was added with 500 μl ECM with 10% exosome‐free FBS. After 4 or 6 h, cells on the lower surface of the chamber were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet and counted. The μ‐Slide Angiogenesis (81506, ibidi) was used for the tube formation assay. HUVECs (1 × 104 cells) in ECM with 2% FBS were seeded on the μ‐Slide that was prechilled and coated with 10 μl of Matrigel (356234, BD). After 3 h, the total tubes were quantified by image analysis of ImageJ.
4
Comprehensive Cellular Functional Assays
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After being transfected with siRNA for 24 h, the cells were seeded into 96-well plates. The CCK-8 kit (YEASEN, 40203ES76) was employed following the manufacturer's instructions. The absorbencies at 450 nm were measured by spectrophotometer reader (Thermo Fisher Scientific, USA)
Cells were seeded in 6-well plates (1000 cells per well) and cultured for 14 days. Colonies were fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using ImageJ software.
The 24-well transwells (8 µm pore size, Falcon, USA) were used for cell migration and invasion assays. 5 × 104/200 µL cells were cultured in a serum-free medium in the upper chambers, and a medium containing 10 % FBS was added to the lower chambers. Matrigel (Corning, USA) was added for cell invasion assays. After 16–24 h, cells adhered to the bottoms of the upper chambers were fixed, stained and counted. Migration and invasiveness were determined by counting cells in five random fields.
3 × 104 cells were suspended in 70 µL of DMEM medium and seeded in an ibidi culture insert (ibidi, Germany). The incubation chambers were placed in a 12-well plate. When cells were at full confluency, the insert was removed and a serum-free medium was added. The wounds were photographed at 0 and 48 h, and the migration rate of the cells was expressed as relative gap closure using the ImageJ v10 software.
Cells were seeded in 6-well plates (1000 cells per well) and cultured for 14 days. Colonies were fixed with glutaraldehyde (6.0% v/v), stained with crystal violet (0.5% w/v) and counted using ImageJ software.
The 24-well transwells (8 µm pore size, Falcon, USA) were used for cell migration and invasion assays. 5 × 104/200 µL cells were cultured in a serum-free medium in the upper chambers, and a medium containing 10 % FBS was added to the lower chambers. Matrigel (Corning, USA) was added for cell invasion assays. After 16–24 h, cells adhered to the bottoms of the upper chambers were fixed, stained and counted. Migration and invasiveness were determined by counting cells in five random fields.
3 × 104 cells were suspended in 70 µL of DMEM medium and seeded in an ibidi culture insert (ibidi, Germany). The incubation chambers were placed in a 12-well plate. When cells were at full confluency, the insert was removed and a serum-free medium was added. The wounds were photographed at 0 and 48 h, and the migration rate of the cells was expressed as relative gap closure using the ImageJ v10 software.
5
Cell Invasion Assay Using Transwell
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Cell invasion assays were performed using 24-well transwells (8-μm pore size; BD Biosciences), coated with Matrigel (Falcon354480; BD Biosciences). RCC cells were starved overnight in serum-free medium, trypsinized, and washed 3 times in DMEM containing 1% FBS. A total of 1 × 105 cells was then suspended in 500 μl DMEM containing 1% FBS and added to the upper chamber, while 750 μl DMEM containing 10% FBS was placed in the lower chamber. For the control, medium containing 1% FBS was added to the lower chamber. After 24 hours of incubation, Matrigel and cells remaining in the upper chamber were removed by cotton swabs. Cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet. Cells in at least 6 random microscopic fields (magnification, × 100) were counted and photographed. All experiments were repeated 3 times.
6
Matrigel-Based Cell Invasion Assay
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For cell invasion assay, the 24-well transwells (8 mm pore size; BD Biosciences, San Jose, CA), coated with Matrigel, were used. A total of 1 × 105 cells were suspended in serum-free medium in the top chamber, while medium containing 10 mg fibronectin and 10% FBS was added into the bottom chamber. After incubated for 48 hours, the translocated cells were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet and observed under a microscope. For quantification, the average number of invasive cells in five fields (left, right, upper, lower, and middle) was applied.
7
Neutrophil Chemotaxis Assay Protocol
8
High-throughput Transwell Migration Assay
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After starvation for 24 h (0.1% FBS), 5 × 104 cells were added to the top chamber of 24-well transwells (BD Bioscience, Bedford, MA, Cat#353097), and 10% FBS assay medium was added to the bottom chambers and incubated for 24 h. After non-migratory cell removal, membranes were fixed, stained with 4′,6-diamidino-2-phenylindole and imaged using a Zeiss Axio Observer Z1 Inverted Epi-fluorescence microscope.
For the screen, 6.6 × 105 starved MDAMB231 cells were plated onto 6-well transwell chambers (BD Bioscience, Bedford, MA, Cat# 353093). After a 24-hour incubation, the non-migratory cells were collected, propagated, and allowed to re-migrate for enrichment purposes. Cells from 8 transwells were combined per cycle to ensure a >700 library coverage. Simultaneously, migration in each cycle was determined in 24-well plates as described before.
For the screen, 6.6 × 105 starved MDAMB231 cells were plated onto 6-well transwell chambers (BD Bioscience, Bedford, MA, Cat# 353093). After a 24-hour incubation, the non-migratory cells were collected, propagated, and allowed to re-migrate for enrichment purposes. Cells from 8 transwells were combined per cycle to ensure a >700 library coverage. Simultaneously, migration in each cycle was determined in 24-well plates as described before.
9
VEGF/VEGFR2 Signaling and GESTEC Migration
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To investigate the effects of VEGF/VEGFR2 signaling pathway on the migration of GESTECs, we performed an in vitro cell migration assay using a 24-well transwell with a pore size of 8 μm (BD Biosciences, Franklin Lakes, NJ, USA). MDA-MD-231/Luc cells (1 × 105 cells/well) were cultured in the lower chamber of the transwell and fibronectin was applied to the transwell to induce the adhesion of migrated GESTECs. To inhibit VEGF/VEGFR2 signaling, stem cells was exposed to KRN633 (Selleckchem, Houston, TX, USA) diluted media before being seeded in the transwell. Briefly, after starvation in serum free media, 100 μM KRN633 was applied to each stem cell for 1 h. The CM-DiI pre-stained GESTECs (1 × 105 cells/well) were then seeded in the upper chamber of transwell after treatment with KRN633. The chamber was subsequently incubated at 37°C for 24 h. After incubation, the non-migrated cells were scraped with a plastic blade and then fixed in cold methanol. The upper chambers of transwell were washed with PBS and stained with 200 ng/ml 4′, 6-Diamidino-2-phenylindole (DAPI; Sigma-Aldrich Co.). Finally, migrated cells were examined with an Olympus microscope (IX71 Inverted Microscope, Olympus, Japan) connected to a fluorescence detector.
10
Quantification of Cell Migration and Invasion
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Migration assay was conducted with ibidi® culture insert. Cell was seeded at both side of the insert (35,000 cells in 70 μl medium) and incubated overnight. The insert was removed next day and photographed at 0 and 48 h. The gap between cells was quantified with Image J and presented as percentage of closure compared to 0 h.
The invasion assay was described previously [28 (link)]. Briefly, 100 μl of 80 μg Matrixgel (BD) was previously loaded onto the upper chamber of 24 well transwell (BD, 8 μm pore size) at 37 °C for 2 h and 5 × 104 cells were seeded on the gel (in 200 μl of medium without FBS) and 500 μl of complete medium was added into the lower chamber of the transwell. Cells invaded through transwell were stained after 24 h incubation. Five images were photographed for each transwell under 100X magnification. Cell numbers were counted and calculated.
The invasion assay was described previously [28 (link)]. Briefly, 100 μl of 80 μg Matrixgel (BD) was previously loaded onto the upper chamber of 24 well transwell (BD, 8 μm pore size) at 37 °C for 2 h and 5 × 104 cells were seeded on the gel (in 200 μl of medium without FBS) and 500 μl of complete medium was added into the lower chamber of the transwell. Cells invaded through transwell were stained after 24 h incubation. Five images were photographed for each transwell under 100X magnification. Cell numbers were counted and calculated.
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