Migration assays were performed using 24-well Transwell® culture chambers (Costar, Cambridge, MA). Lower chambers were filled with DMEM containing 5% FBS. Equal numbers (5×104) of human melanoma cell lines (G361, A375, SK-MEL-5, SK-MEL-28, Hs 294T and WM-266-4) or transfectants (G361-vector and G361-IL-32α) were added to the upper insert with serum-free DMEM. Transwell chambers were incubated at 37°C in a 5% CO2 humidified incubator for 24 hours. After incubation, migrated cells were fixed with methanol and stained with crystal violet solution. After imaging, the stained cells were eluted in 10% acetic acid and optical density (O.D.) values at 570 nm were measured using an ELISA reader (Molecular Devices, Sunnyvale, CA). The migratory ability of cells was assessed in triplicate wells.
Transwell culture chamber
Manufactured by Corning
Sourced in United States
Transwell culture chambers are a type of lab equipment used for cell culture experiments. They consist of a two-compartment system with a semi-permeable membrane insert that allows for the study of cell interactions and the transport of molecules across a cellular barrier.
Automatically generated - may contain errors
Lab products found in correlation
Elisa reader, by Molecular Devices (2 mentions)
Matrigel, by Corning (2 mentions)
Matrigel matrix, by BD (2 mentions)
Fluorescence microscope, by Leica (1 mentions)
Matrigel, by Leica (1 mentions)
Optical microscope, by Zeiss (1 mentions)
Crystal violet, by Genview (1 mentions)
Crystal violet, by Solarbio (1 mentions)
Matrigel coated, by BD (1 mentions)
Fibronectin, by Roche (1 mentions)
Matrigel, by BD (1 mentions)
Mitomycin c, by Nacalai Tesque (1 mentions)
Spectrafluor plate reader, by Tecan (1 mentions)
Optical microscope, by Leica (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Methanol, by Solarbio (1 mentions)
16 protocols using transwell culture chamber
1
Melanoma Cell Migration Assay
2
Transendothelial Migration Assay for MCF-7 Cells
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A transendothelial migration assay was performed to detect the MCF-7 cells that invaded through HUVEC monolayers without or with Fx treatment (Zhang et al., 2020 (link)). Briefly, the transwell culture chambers (24-well, 8 μm pore size, Costar, Corning, United Statws) were placed in a 24-well plate, the upper chambers were coated with Matrigel (Corning, United Statws, Cat. #356234) on the bottom and allowed to air-dry under sterile conditions. HUVECs (1 × 105/well) were seeded in the upper chamber and allowed to grow to confluence. Then, the addition of TNF-α (10 ng/ml) to stimulate the monolayer of HUVECs for 4 h. The medium was removed, and GFP-labeled MCF-7 cells (5 × 104/well) were suspended in 500 μl of serum-free medium containing various concentrations of Fx (0 μM, 5 μM, 10 μM, and 25 μM) and placed into the upper chamber of the wells. 500 μl of medium containing 20% FBS and the same concentration of Fx was added to the lower chambers. After a further 24 h of incubation at 37°C in 5% CO2, the cells in the upper chambers were carefully wiped with a cotton swab and washed with PBS. The fluorescence signal of the MCF-7 cells (labeled with GFP) that transendothelial migrated to the basolateral side of the Matrigel coated-transwell membrane was recorded by using a fluorescence microscope (Leica, Germany).
3
Cell Invasion Assay Protocol
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The invasion assay was performed using transwell culture chambers (8 µm pores; Costar, Corning, NK, USA) according to the manufacturer's instructions. The upper chamber was loaded with 1×105 cells in 0.2 ml serum-free medium, while 0.6 ml medium containing 10% FBS was loaded to the lower chamber. After incubation for 48 h at 37°C in a humidified atmosphere of 5% CO2, the cells on the lower side were fixed in 95% ethanol and stained with crystal violet at room temperature for 10 min and counted under a microscope (Olympus Corp.). Four microscopic fields were randomly selected for cell counting. The images were captured at ×100 magnification. Each experiment was performed at least three times.
4
Transwell Migration Assay for Synovial Fibroblasts
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Migration assays were performed using 24-well Transwell® culture chambers (Costar, Cambridge, MA). Lower chambers were filled with Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS). Equal numbers (3×104) of SW982 human synovial fibroblasts were added to the upper insert in serum-free DMEM. Transwell chambers were incubated at 37°C in a 5% CO2 humidified incubator for 24 h. After incubation, cells that had migrated to the underside of the membrane were fixed in methanol and stained with crystal violet. After imaging, stain was eluted from the cells in 10% acetic acid, and optical density (O.D.) at 570 nm was measured using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA). The migratory ability of cells was assessed in triplicate wells.
5
Transwell Invasion and Migration Assay
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For the transwell invasion and migration assay, we prepared transwell culture chambers (24-well, 8 μm pore size, Costar, Corning Incorporated, USA) coated with/without 60 μL Matrigel (Corning, TewksBury, MA). After incubation at 37°C for 2 hrs, the transfected or control cells (1×105 cells) in 200 μL RPMI (0.1% BSA）were added into the upper compartment of the chambers, and 800 μL medium containing 20% FBS was used as the attractant and placed in the bottom chambers. After incubation for 48 hrs at 37°C and 5% CO2, non-invaded cells on the upper chamber were scraped off with a cotton swab. The invaded cells on the lower membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (genview) for 30 mins. The invading cells were counted and photographed (five random fields) by using optical microscope (Zeiss, Germany).
6
Transwell Assay for Chemotactic Migration
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The chemotactic cell migration was measured using 24-well Transwell culture chambers (Costar; 8-mm pore size). The upper chamber was coated with gelatin B (1 mg/ml) and air-dried for 1 h. HASMCs were transfected with the miRNA inhibitors for 24 h, serum-starved for 18 h, and re-plated on the upper chambers with basal media at a density of 6000 cells/chamber. Complete media were added to the lower chambers. Transwell chambers were incubated at 37 °C for 24 h. The non-migrated cells were removed from the upper side of the membranes, and the cells passed through and attached to the lower side of the membranes were fixed and stained with 0.6% hematoxylin and 0.5% eosin. The number of stained cells from 4 different fields was counted and averaged.
7
Transwell Assay for Cell Migration
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Cell migration was assessed using 24-well Transwell culture chambers (Corning, Corning, NY, United States). Cells (2 × 104) suspended in 200 µl serum-free medium were seeded in the upper chamber. The lower chamber was filled with 800 µl complete medium. After incubation at 37°C for 24 h, the cells were fixed in 4% paraformaldehyde for 20 min. After being washed thrice with PBS, the cells were stained using 1% crystal violet (Solarbio, Beijing, China) for 10 min. Cells that transmigrated to the lower chamber were counted. Data were obtained from at least three independent experiments.
8
Transwell Migration Assay with Eupatilin
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Transwell assay was performed using Transwell® culture chambers (Corning Life Sciences, Corning, NY, U.S.A.). The lower chamber was filled with 500 μl DMEM containing 10% FBS with TGF-β1 (10 ng/ml). ASMCs (1 × 105) resuspended in 500 μl serum-free DMEM were placed in the upper chamber. TGF-β1 (10 ng/ml) was added to the cells with or without the presence of eupatilin (10, 20, 40 μM), and incubated for 24 h. After removing the non-migrated cells on the upper side of the inserts using a cotton swab, the migrated cells on the lower side of the inserts were fixed using 95% methanol for 10 min and stained with 0.5% Crystal Violet for 30 min. The average cell number from five random fields was calculated.
9
Cell Migration and Invasion Assays
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Migration and invasion assays were performed using uncoated or Matrigel-coated (BD Biosciences) transwell culture chambers (8.0-µm pore size; Corning Inc., Corning, NY, USA) according to the manufacturer’s instructions. First, the transwell culture chambers were placed onto 24-well plates. For migration assays, the cells were resuspended in serum-free medium (8×105 cells/mL for Bel-7402 cells, 4×105 for Sk-hep-1 cells), and 100 µL of the suspension was added to the upper chambers. For invasion assays, the cells were resuspended in serum-free medium (10×105 for Bel-7402 cells, 8×105 for Sk-hep-1 cells), and 100 µL of the suspension was added into the upper chambers, which were coated with 50 µL (1.25 mg/mL) of BD Matrigel™ Matrix (BD Biosciences). The lower chambers were both filled with 400 µL of RPMI-l640 or DMEM containing 15% FBS. Following a 24-hour incubation for the migration assay and a 48-hour incubation for the invasion assay, the cells on the lower surface of the upper chambers were fixed in 4% paraformaldehyde, stained with 0.25% crystal violet for 20 minutes, and counted in five random fields at a magnification of 100×. Each experiment was independently repeated in triplicate.
10
Evaluating Cancer Cell Invasion Potential
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Invasiveness of CRC cell line SNU‐1033 was determined by the assay using 24‐well Transwell culture chambers (Corning, Tewksbury, MA, USA) as described previously,8 with some modifications. Briefly, the lower and upper surfaces of filters with an 8.0 μm pore size were coated with 1 μg fibronectin (Roche Diagnostics) and 5 μg Matrigel (BD Biosciences, San Jose, CA, USA), respectively. A total of 1 × 105 cells in 100 μL culture medium were added to the upper compartment of the chamber, and 600 μL culture medium was added to the lower chamber. To exclude any influence of cell proliferation on invasiveness, 50 ng/mL proliferation inhibitor mitomycin C (Nacalai Tesque, Kyoto, Japan) was applied to the culture medium in both upper and lower chambers. After incubation for 24 hours at 37°C in 5% CO2, the filter was fixed with 30% methanol and stained with 0.5% crystal violet in 20% ethanol. The non‐invading cells on the upper surface of the filter were then removed using a cotton swab. Subsequently, the filter was excised from the chamber using a scalpel and the number of invaded cells was counted using a microscope in three predetermined fields at a magnification of ×200.
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