Transwell chambers with 8-μm pore filters (Corning International, Tokyo, Japan) were used to determine the migration ability of MSCs. The upper chambers were plated with 2×104 cells in 200 μL α-MEM containing 0.1% bovine serum albumin (BSA), and the lower chambers with 600 μL VLA-4 (1 μ/μL, Biofine, Beijing, China) in 5% CO2 at 37°C for 10 h. The migrated MSCs on the lower side of the filter were stained by crystal violet and then counted under a microscope.
Transwell chambers with 8 μm pore filters
Manufactured by Corning
Sourced in United States
Transwell chambers with 8-μm pore filters are a type of cell culture insert designed for a variety of cell-based assays. These chambers feature a semi-permeable membrane with 8-micron pores that allow for the passage of cells, molecules, and other materials between the upper and lower compartments of the device.
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8 protocols using transwell chambers with 8 μm pore filters
1
Transwell Assay for MSC Migration
2
Evaluating Cell Proliferation and Migration
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Cell proliferation was evaluated by WST-8 hydrolysis using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.) following the manufacturer’s specifications. HC-OAs were seeded onto 96-well tissue culture plates at 5000 cells per well. Plates were incubated for 24, 48 or 72 h at 37°C with 5% CO2 in a humidified incubator. A total of 10 μL of CCK-8 reagent was added to each well and incubated for 3 h. Absorbance readings were then taken at 450nm using a microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). The migration of EV-treated chondrocytes was assessed using transwell chambers with 8 μm pore filters (Corning Inc., Corning, NY, USA). Briefly, 5 × 104 cells in 100 μL of low serum culture medium (DMEM-F12 supplemented with 0.5% FBS and 1% P/S) were seeded in the upper chamber, and EVs or vehicle control (PBS) was added to the lower chambers. Following incubation for 16 h at 37°C, non-migrating cells were removed with cotton swabs. Cells on the underside of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet solution for 15 min. Stained cells that migrated through the pores to the underside of the membrane were visualised under a microscope and counted at 10× magnification.
3
Invasion Assay of Colon Cancer Cells
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Invasion rates of colon cancer cells with MON1B interference were assessed and compared to Control and NC groups using 24-well Transwell chambers with 8-μm pore filters (Corning, USA) coated with Matrigel GFR (BD, USA). First, 5×104 cells were inoculated in the upper chambers filled with DMEM culture media without FBS. The lower chambers were filled with DMEM culture media containing 10% FBS. After being incubated for 48 h, the lower membrane was fixed with 4% paraformaldehyde (PFA), stained with 0.1% crystal violet for 30 min at room temperature, and then rinsed by 10% acetic acid. The invaded cells were counted under a DMi8 optical microscope (Leica, Germany).
4
Transwell-Based Cell Invasion and Migration Assay
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Cell invasion and migration were analyzed using the Transwell chambers with 8-μm pore filters (Corning, Corning, NY, USA) [16 (link)]. Briefly, the transwell inserts were pre-coated with 0.5-mm thickness of Matrigel (BD Biosciences, Bedford, MA, USA) for cell invasion assay, but not for cell migration. Then, cells were adjusted to 1 × 104 cells/mL and seeded into the upper chamber. The culture medium was added to the low chamber of the Transwell System the culture medium. Approximately 48 h later, the invaded or migrated cells at the bottom surface of filter were fixed with 4% paraformaldehyde, and then stained with 0.1% crystal violet solution. An optical microscope was used to count the number of cells by selecting five random microscopic fields. All experiments were conducted in triplicate.
5
Arsenite-Induced Cell Migration and Invasion
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Migration of arsenite-transformed L-02 cells was evaluated by use of Transwell chambers with 8-μm pore filters (Corning Inc., Corning, NY, USA). At 24 h after transfection, cells (5 × 104/100 μL) were plated on the upper chambers in serum-free medium; 1640 medium containing 10% FBS was added to the lower chambers as a chemoattractant. After incubation for 24 h at 37 °C, non-migrating cells were removed with cotton swabs. Cells that migrated to the bottom of the membrane were fixed with 4% paraformaldehyde, stained with crystal violet solution for 30 min, and washed twice with PBS. Stained cells were visualized under a microscope (high-power field), and the numbers of cells counted in five random fields were averaged. To assess the capacity for invasion of arsenite-transformed L-02 cells, transfected cells (5 × 104/100 μL) were added to upper chambers that had been coated with 35 μL of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA); 1640 medium containing 10% FBS was added to the lower chambers. Cells were incubated for 24 h at 37 °C, and non-invading cells were removed with cotton swabs. Invading cells were fixed, stained, and counted.
6
Transwell Assay for Endothelial Cell Migration
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Transwell chambers with 8 μm pore filters (Corning, Corning, NY, USA) were precoated with 1 mg/ml Matrigel (BD Biosciences, San Jose, CA, USA). HUVEC (5 × 104 cells of each chamber) were starved overnight and added to the upper chamber, and the lower chamber was filled with complete medium with VEGF (or together with P31). After 24 h, cells were immersed into 3 % paraformaldehyde for 15 min, stained with crystal violet, and counted under a light microscope [29 (link)].
7
Evaluation of CXCL14 Knockdown on MCL Cell Migration
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MCL cells were treated as follows: control group, scramble group, and siCXCL14 group. After transfection of 48 h, 2 × 106 cells/mL of each cell type was starved in serum-free 1640 for 12 hours at 37°C in 5% CO2. Migration assays were subsequently performed using Transwell chambers with 8-μm pore filters (Corning, Corning, NY, USA). Cell suspensions (2 × 105 in 100 μL) were added to the upper chambers and 600 μL of medium either containing 10% FBS was added to each of the lower chambers. After transwells were incubated for 24 hours at 37°C in 5% CO2, the cells in each lower chamber were recovered and counted using CCK8 assay; the entire assay was repeated 3 times.
8
SUMO2 Impacts Cell Invasion
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Cell invasion assays were performed using Transwell chambers with 8‐μm pore filters (Corning, New York, NY, USA). SMMC‐7721 and Bel‐7404 cells were seeded in six‐well plates and transfected with siSUMO2 or NC and incubated for 36 h. The cells were then seeded at a density of 5 × 104 cells per well in 200 μL of serum‐free DMEM in the upper chamber; the bottom chamber contained 600 μL of DMEM with 10% FBS as a chemoattractant. After a 24‐h incubation, cells were fixed with methanol and stained with 0.1% crystal violet. Five random fields were photographed at ×200 magnification using a digital microscopic imaging system (Leica, Bensheim, Germany), and the cells within these fields were counted.
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