Cells were trypsinized at 48 h post-transfection, resuspended, and adjusted to a concentration of 2 × 10 5 cells/mL in serum-free culture medium. Transwell invasion chambers (BD Biosciences, San Jose, CA, USA) were placed into 24-well plates, and 500 µL of RPMI-1640 culture medium containing 10% FBS was then added to the lower chamber, while 200 µL of cell suspension was added to the upper chamber. The Transwell chamber was collected after 24 h of culture, and a cotton swab was used to wipe cells from the upper chamber. The back of the chamber was washed by immersion in phosphate-buffered saline, fixed with 4% paraformaldehyde in PBS, and stained with 0.1% crystal violet. Using an optical microscope (Olympus Corporation, Tokyo, Japan), six different fields of view were randomly selected for imaging, and the number of migrating cells within the fields were counted. For the cell invasion experiment, Transwell invasion chambers were first covered with 20 µL of 0.5 g/L Matrigel (BD Bioscience) artificial matrix and incubated at 37°C for 4 h. After gel formation, cells were inoculated onto the chambers for 48 h. The subsequent experimental procedures were identical to those described for the cell migration experiments.
Transwell invasion chambers
Manufactured by BD
Sourced in United States
Transwell invasion chambers are laboratory equipment designed to assess the ability of cells to migrate through a barrier. The chambers consist of an upper compartment separated from a lower compartment by a membrane. Cells are seeded in the upper compartment and their ability to invade and migrate through the membrane is measured, providing insights into cellular behavior and function.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (6 mentions)
Bulletkits, by Lonza (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
3 3 diaminobenzidine dab peroxidase substrate kit, by Vector Laboratories (1 mentions)
Tgf β1 elisa kit, by Thermo Fisher Scientific (1 mentions)
22rv1 cell, by American Type Culture Collection (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lncap, by American Type Culture Collection (1 mentions)
Cas block, by Thermo Fisher Scientific (1 mentions)
Silibinin, by Merck Group (1 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
Transwell migration chamber, by Corning (1 mentions)
Mtt cell viability assay, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Dmem f12 serum free medium, by Thermo Fisher Scientific (1 mentions)
Matrigel, by Merck Group (1 mentions)
12 protocols using transwell invasion chambers
1
Cell Invasion and Migration Assay
2
Prostate Cancer Cell Line Characterization
3
Cell Migration and Invasion Assay
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Cell migration was assessed by wound-healing assay. An artificial wound was scratched on a confluent cell monolayer without FBS using sterile tips, and wound-healing images were taken at 24 and 48 h later. Cell invasion was assessed using transwell invasion chambers coated with matrigel (BD Biosciences, Franklin Lakes, NJ, USA). 0.2 ml of cells suspended in serum-free medium was added into the upper chamber. The lower chamber was filled with 500 ul of RPMI 1640 or DMEM medium with 10 % FBS as the nutritional attractant. 24 h later, cells remaining on the upper side of the membrane were removed, and cells that migrated through the membrane were fixed with 75 % alcohol and stained with crystal violet, and the invasive cells were counted and imaged using an inverted microscope (Nikon, Japan).
4
Investigating Cancer Cell Invasion Inhibition
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TABLE 7 Invasion assay results % of Cells Migrated Compared Compound to Control GW3965 <70% SB742881 <70% WO20100138598 Ex. 9 <90% DMSO Control 100% 1 <95% 8 <50% 9 <50% 29 <50% 31 <70%
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Example 85
Cancer cells (MeWo) were pre-treated in presence of 1 μM drug or control (DMSO) for 72 h after which cancer cells were switched to serum-starvation in 0.2% FBS DMEM-based media for 16 h in the presence of the drug or control, respectively. Trans-well invasion chambers (354480, BD Biosciences, Bedford, Mass.) were pre-equilibrated in DMEM-based media prior to the beginning of the assay by adding 0.5 ml of starvation media to the top and bottom chambers. After 30 min, the media in the top chamber was removed, and 0.5 ml of media containing 1×105 cancer cells in the presence of the drug, was added into each matrigel-coated trans-well insert. Cells were allowed to invade through the matrigel-coated inserts for 24 h at 37° C.
Results: The results of the invasion assay with select compounds are shown in Table 7.
5
Transwell Migration and Invasion Assay
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The migration and invasion abilities of OS cell lines were evaluated using Transwell migration chambers (Costar, USA) and Transwell invasion chambers precoated with 50 μl of 2 mg/ml Matrigel (BD Biosciences, USA), respectively. In brief, transfected cells (4 × 104 cells/well for the migration assay, 8 × 104 cells/well for the invasion assay) suspended in 200 μl of serum-free DMEM were seeded into the upper chambers. A 600 μl volume of DMEM supplemented with 10% FBS was used as the attractant and was added into the lower chambers. After culture for 24 h, cells adhering to the lower surface of the membrane were fixed with paraformaldehyde (4%) and stained using crystal violet (0.1%), whereas cells on the upper surface of the membrane were removed by wiping with cotton swabs. At least three random fields of view containing cells that had migrated or invaded to the lower surface were imaged under an inverted light microscope.
6
Transwell Invasion Assay for Fibroblasts
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Transwell invasion chambers (membrane pore size, 8 μm) coated with Matrigel (BD Biosciences) were placed into 24-well plates. Following an overnight culture in serum-free medium, KFs (1×105 cells/well) were added to the upper chambers and were incubated with or without various Rg3 concentrations (50 or 100 μg/ml) for 24 and 48 h. Normal medium containing serum was placed into the lower chambers. After 24 or 48 h, cells that remained on the upper surface of the membrane were completely removed using a cotton swab. Cells that crossed the Matrigel and migrated to the lower side of the Transwell insert were fixed with 4% paraformaldehyde for 5 min at room temperature and stained with DAPI. The number of cells that invaded across the membrane was counted in five random fields under an Olympus CX40 fluorescence microscope (olympus Corporation).
7
Transwell Invasion Assay of Glioma Cells
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Transwell invasion chambers (BD Biosciences) were used according to the manufacturer’s instructions, and 100 μl collagen mix was used to coat the membrane. Adenovirus infected U251, U138, T98G and naïve U251 cells were plated onto a transwell membrane insert at a density of 1 × 105 cells per well in 100 μl DMEM. The lower chambers were filled with 500 μl DMEM. The transwells were then incubated for 24 h to allow cells to migrate. At the end of incubation, cells on the upper side of the insert filter were completely removed using a cotton swab, and cells that had invaded through the collagen-coated membrane were fixed in 4% PFA and stained with hematoxylin and eosin. For quantification, cells were counted under a microscope in eight random fields at 20×magnification. The experiment was repeated in triplicate.
8
Quantifying Cell Migration and Invasion
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Cells were seeded into 35 mm dishes and treated accordingly before being trypsinized, resuspended in 0.1% FCS-containing DMEM and seeded in equal quantities into 12-well Transwell migration chambers (Greiner Bio-One, Austria) or 24-well matrigel covered Transwell invasion chambers (BD Biosciences, USA) with an 8 μm pore size. The chambers were placed into a lower chamber containing 20% FCS-containing DMEM and the cells allowed to migrate/invade through the membrane or matrigel matrix over 24 hrs. The cells that were unable to move through the membrane were removed while the remaining cells were fixed in methanol, stained with crystal violet, counted and imaged using a Zeiss Primovert inverted phase microscope. Results were normalized to an MTT cell viability assay (according to the manufacturer's protocol, Sigma-Aldrich, USA) and western blotting confirmed KPNB1 knockdown.
9
Quantifying Cancer Cell Invasion and Stemness
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Cell invasion and tumorsphere assays were performed as previously described [72 (link),73 (link)]. For invasion assay, the cells in DMEM with 1% FBS were seeded into Transwell invasion chambers (BD Biosciences) coated with Matrigel (Merck). The lower chambers contained 10% FBS to trap cells. After 16 h, the cells invading the reverse side of the membrane were fixed, stained, and photographed. For tumorsphere assay, the cells were seeded on ultra-low attachment plates (Merck, Darmstadt, Germany) in the DMEM/F-12 serum-free medium (Gibco, Waltham, MA, USA) with B27 supplement (Invitrogen) and growth factors bFGF and EGF. The plates were incubated at 37 °C for 10 days. Cell spheres were visualized and enumerated using light microscopy.
10
Transwell Invasion Assay Protocol
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Invasion assays were performed in triplicate using Transwell invasion chambers coated with Matrigel (50 μl per filter) (BD Biosciences, Franklin Lakes, NJ, USA) in accordance to the manufacturer's protocol. After being cultured for 48 h, cells were transferred on the top of Matrigel-coated invasion chambers in a 1% fetal calf serum DMEM/F12 (2 × 104 cells/well). The lower chambers were added with DMEM/F12 containing 10% fetal calf serum. Cells were incubated for 24 h at 37°C in an atmosphere containing 5% CO2. Subsequently, invaded cells on the lower surface were stained with crystal violet stain and counted under a light microscope.
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