In our study, the invasion of cancer cells was analyzed using the Transwell assay. Approximately 5×105 cells were plated in the upper chambers of 8 µm transwell inserts (BD Biosciences, USA) in serum-free media. Then the lower chambers were filled with normal growth medium. The cells were incubated for 24 hrs allowing for invasion. Cells that migrated to the lower chambers were washed with PBS, mixed with 20% methanol and stained with 0.1% crystal violet. This assay was repeated three times, and the number of stained cells in five randomly chosen fields was determined under a microscope.
8 m transwell inserts
Manufactured by BD
Sourced in United States
The 8-μm Transwell inserts are a laboratory equipment used for cell culture applications. They feature a porous membrane with 8-micron pores that allow for the controlled migration and invasion of cells across the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (4 mentions)
Crystal violet, by Keygen Biotech (4 mentions)
Paraformaldehyde (pfa), by Solarbio (3 mentions)
Dmi4000b, by Leica (3 mentions)
Inverted microscope, by Nikon (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Dtx500, by Nikon (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Ts100 f, by Nikon (1 mentions)
11 protocols using 8 m transwell inserts
1
Transwell Invasion Assay for Cancer Cells
2
Transwell Invasion Assay for Cells
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Transfected cells (5×104 cells/well) were resuspended in serum-free DMEM and seeded into the upper chamber of 8-µm Transwell inserts (BD Biosciences), which had been pre-coated with Matrigel (BD Biosciences) at 37°C for 30 min. The lower chamber was plated with DMEM supplemented with 10% FBS, and the cells were incubated at 37°C for 24 h. The cells were then fixed using 4% paraformaldehyde at room temperature for 30 min, and then stained using crystal violet at room temperature for 5 min, before being imaged under a light microscope (magnification, ×100). The number of invading cells was counted in five random non-overlapping fields.
3
Transwell Assay for Cell Invasion and Migration
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Cell invasion and migration were assessed using a transwell assay. For cell invasion experiment, 2×105 MDA-MB-231 cells transfected with different plasmids were trypsinized into a single-cell suspension and placed on top of 8 µm transwell inserts (BD Biosciences, San Jose, CA, USA) precoated with Matrigel basement membrane matrix (BD Biosciences). Additionally, the lower chamber was supplied with 600 µL of culture media containing 20% FBS as a chemoattractant, followed by an incubation at 37°C with 5% CO2 for 24 h. Subsequently, the cells remaining on the apical side of each transwell membrane were carefully scraped off with a cotton swab, while cells that had invaded to the other side of the membrane were fixed with 4% paraformaldehyde at room temperature for 30 min, stained with 0.1% crystal violet for 10 min, washed with PBS three times, and dried at 80°C for 30 min. Finally, the number of invasive cells was counted from 4 to 5 randomly microscopic filed under an inverted microscope (×400 magnification; Nikon Corporation, Tokyo, Japan). However, for cell migration experiment, the concentration of MDA-MB-231 cells in each group was adjusted to 1×105 cells/well and similarly placed on top of 8 µm transwell inserts but without Matrigel basement membrane matrix and the other procedures were similar to invasion experiment.
4
Evaluating GLSE's Impact on Cell Migration
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The effect of GLSE on the trans-migration of UW-BCC1 and A431 cells was analyzed using a trans-well migration assay using 8-µm trans-well inserts (BD Bioscience). UW-BCC1 and A431 cells (5 × 105) were suspended in culture media containing GLSE (0–160 μg/mL) and reduced FBS (1%), and were seeded onto 8 μm pore size polyethylene terephthalate (PET) membranes. Appropriate culture media were then supplemented with 10% FBS and added to the bottom of each well and incubated for 48 h. After incubation, the cells remaining on the upper membrane were removed by cotton swabs, whereas the cells that migrated to the bottom of the PET membrane were fixed and stained with 0.5% gentian-violet as above, air-dried and counted. The number of cells that passed through the membranes was quantified by counting 20 random fields under light microscopy at 100× magnification. Each condition was tested in three separate wells from three independent experiments. For quantification, gentian violet was dissolved in 50% acetic acid and absorbance at 540 nm was measured.
5
Transwell Invasion Assay for SiHa and HeLa Cells
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The stable cell lines SiHa siRNA-NC, SiHa siRNA393, SiHa siRNA613, HeLa siRNA-NC, HeLa siRNA393 and HeLa siRNA613 were counted and then 10 × 104 stably infected SiHa cells and 20 × 104 stably infected HeLa cells in 250 µl of serum-free medium were separately plated into the upper chamber of 8-µm transwell inserts (BD Biosciences, Franklin Lakes, NJ), while 500 µl of medium containing 10% bovine serum albumin was added to the lower chamber. After 24 h of incubation at 37 °C, SiHa siRNA cells in the upper chamber were removed carefully. After 48 h of incubation at 37 °C, HeLa siRNA-NC and HeLa siRNA cells in the upper chamber were removed. Migrated cells on the lower membrane surface were fixed in 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min and then stained with 0.1% crystal violet (KeyGEN biotech, Nanjing, China) for 10 min. The number of cells was counted in 5 randomly selected visual fields (100×) per well under an inverted microscope DMI4000B (Leica, Wetzlar, Germany).
6
Matrigel Invasion Assay for Glioma Cells
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In vitro analysis of invasion was assessed using Matrigel (BD Biosciences, Frankling Lakes, NJ, USA) and 8-µm Transwell inserts (BD Biosciences). The cell invasion assay was performed as previously described (18 (link)). Briefly, 50 µl Matrigel (diluted 1:5 with serum-free DMEM) was plated onto the Transwell insert. Then a cell suspension of 5×103 U251MG or U-87MG ATCC cells was added. Treatments of 10 µM apatinib and 20 µM TMZ were made for 24 h at 37°C. The invasive cells were fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) and stained with 10% crystal violet (Beyotime Institute of Biotechnology) at room temperature (from 22 to 25°C) for 15 mins. The invasive cells were photographed by light microscopy (DTX500; Nikon Corporation, Tokyo, Japan) with a magnification of ×40 and the cells in three random fields of view were counted.
7
Cell Migration and Invasion Assays
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Cells were seeded in a 12-well plate and then transfected with siRNA or plasmid or pretreated with C75 (50 µM) and Cerulenin (50 µM) inhibitors for one hour until reaching 100% confluence. A scratch presenting a wound was made inside the well, and then, the cells were cultured for another 48 h to observe cell migration. The closure area was quantified to indicate the cell migration. For Transwell migration/invasion assays, a total of 2 × 104 cells in 200 µl of serum-free medium were seeded in the upper chamber of 8-µm Transwell inserts (BD Biosciences, Franklin Lakes, NJ, USA). The lower chamber was filled with 10% FBS medium. Twenty-four hours later, the remaining cells in the upper chamber were removed. The cells adhering to the membrane were fixed with methanol for 15 min and then stained with 0.1% crystal violet (KeyGEN Biotech, Nanjing City, China) for 30 min. For the invasion assays, the upper chamber was precoated with 50 µl of Matrigel (#356234, BD Biosciences), and 2 × 105 cells in 200 µl of serum-free medium were seeded. The rest of the procedure was the same as that for the migration assays.
8
Transwell Assay for Cell Migration
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The stable cell lines SiHa siRNA-NC, SiHa siRNA393, SiHa siRNA613, HeLa siRNA-NC, HeLa siRNA393 and HeLa siRNA613 were counted and then 10 × 10 4 stably infected SiHa cells and 20 × 10 4 stably infected HeLa cells in 250 µl of serum-free medium were separately plated into the upper chamber of 8µm transwell inserts (BD Biosciences, Franklin Lakes, NJ), while 500 µl of medium containing 10% bovine serum albumin was added to the lower chamber. After 24 h of incubation at 37°C, SiHa siRNA cells in the upper chamber were removed carefully. After 48 h of incubation at 37°C, HeLa siRNA-NC and HeLa siRNA cells in the upper chamber were removed. Migrated cells on the lower membrane surface were xed in 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min and then stained with 0.1% crystal violet (KeyGEN biotech, Nanjing, China) for 10 min. The number of cells was counted in 5 randomly selected visual elds (100×) per well under an inverted microscope DMI4000B (Leica, Wetzlar, Germany).
9
Transwell Assay for Cell Migration
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The stable cell lines SiHa siRNA-NC, SiHa siRNA393, SiHa siRNA613, HeLa siRNA-NC, HeLa siRNA393 and HeLa siRNA613 were counted and then 10 × 10 4 stably infected SiHa cells and 20 × 10 4 stably infected HeLa cells in 250 µl of serum-free medium were separately plated into the upper chamber of 8µm transwell inserts (BD Biosciences, Franklin Lakes, NJ), while 500 µl of medium containing 10% bovine serum albumin was added to the lower chamber. After 24 h of incubation at 37°C, SiHa siRNA cells in the upper chamber were removed carefully. After 48 h of incubation at 37°C, HeLa siRNA-NC and HeLa siRNA cells in the upper chamber were removed. Migrated cells on the lower membrane surface were xed in 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min and then stained with 0.1% crystal violet (KeyGEN biotech, Nanjing, China) for 10 min. The number of cells was counted in 5 randomly selected visual elds (100×) per well under an inverted microscope DMI4000B (Leica, Wetzlar, Germany).
10
Transwell Assay for Cell Migration
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Cell migration assays were assessed using transwell chambers (8 µm transwell inserts, Falcon, Thermo Fisher Scientific). HCC cells in serum-free DMEM were seeded in the upper chamber, and medium containing 10% FBS was added in the lower chamber as the chemoattractant. After 24 or 48 hours of incubation, the penetrated cells on the filters were fixed and stained with 0.1% crystal violet. Migranted cells were counted in five random fields (×10 magnifications) and imaged using SPOT imaging software.
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