Cells were seeded at sub-confluent concentrations (10-20 thousand cells/cm2) on polycarbonate Transwell membranes with pore diameters of 3 μm (Corning, CLS3414), 5 μm (Corning, CLS3421), and 8 μm (Corning, CLS3428). Membranes were pre-coated with collagen 1 (50 ug/mL). After seeding cells, the filters were maintained at 37°C and 5% CO2 for 15 hours. Cells were gently removed from either the top or bottom of the membrane with a cotton swab and immediately fixed with paraformaldehyde. To determine the number of cells per unit area, cells were stained with crystal violet and imaged at multiple locations across the membrane with a 10x objective. Cells were manually counted in 800×800 μm2 fields of view (12-30 locations per condition). The percentage of cells that cross the membrane (Fig. 5 ) was then determined by taking the ratio of the number of cells on the bottom of the membrane and the sum of cells on the filter top and bottom.
Cls3428
Manufactured by Corning
Sourced in United States
The CLS3428 is a laboratory equipment product manufactured by Corning. It serves as a core function for laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
6 protocols using cls3428
1
Cell Migration Across Microporous Membranes
2
Cell Migration Assay with RSC96
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RSC96 cells following completion of transfection were inoculated in the upper chamber (8 μM, CLS3428, Corning, USA) as previously mentioned. In the lower chamber, 500 μL of DMEM medium containing 10% FBS was added. The co-culture was then carried out at 37 °C with 5% CO2 for 12 h. After the co-culture, the cells at the bottom of the upper chamber were wiped away using a cotton swab. The remaining cells were stained with 0.1% crystal violet and imaged. The cell count was performed under a microscope.
3
Motility, Soft Agar, and Invasion Assays
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Random motility was assayed by time-lapse video microscopy as previously described [61 (link)]. For soft agar growth, 0.5 x105 sarcoma cells were seeded in 0.3% top agar and incubated at 37°C. Foci were evidenced with MTT staining and counted by using ImageJ, as previously described [62 (link)]. For invasion assay, each well of the invasion chamber (CLS3428, Corning) was coated with 200μl of Matrigel matrix coating solution (Cultrex, Trevigen). Next, a cell suspension of 0.5x105 LMS cells in 0.1%FBS-DMEM was added. As chemoattractant, 20%FBS-DMEM was added in each lower chamber. As a control 0.1%FBS-DMEM was used to evaluate random invasion.
4
Matrigel-Based Cell Invasion Assay
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Each well of the invasion chamber (CLS3428, Corning, New York, NY, USA) was coated with 200 μL of Matrigel matrix coating solution (Cultrex, Trevigen, Gaithersburg, MD USA). Next, a cell suspension of 3 x104 cells in 0.1 % FBS- DMEM or DMEM low glucose was added. As chemoattractant, 20 % FBS-DMEM was added in each lower chamber. As a control, 0.1 % FBS-DMEM was used to evaluate random invasion.
5
Differentiation and Activation of Bone Marrow-Derived Macrophages
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Primary bone marrow cells were isolated from the femur and tibia of the Moff/f; ER-Cre mice and cultured in DMEM supplemented with 20% L929 conditioned medium, 10% heat-inactivated FBS, 100 nM 4-OHT, 100 units/ml penicillin and streptomycin for 4 days to differentiate into bone marrow–derived macrophages (BMDMs). BMDMs were activated by culturing with 50% hepatocyte-conditioned medium for 18 h. For coculture experiment, 1 × 105 BMDMs were seeded to an insert (Corning, CLS3428) with hepatocytes at bottom for 2 days before the experiment.
6
Cell Invasion Assay Using Matrigel
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The upper chamber (cat. CLS3428, Corning, USA) of a 24-well plate (cat. CLS3527, Corning, USA) was covered with 50 μl Matrigel matrix (cat. 354234, BD, USA) and placed at 4ºC. Then, 200 μl cell suspension at a concentration of 1 × 10 5 cells/ml was introduced to the upper chamber, while 650 μl fresh medium containing 10% FBS was added to the lower chamber as a chemokine. After 24 h of cultivation in an incubator at 37ºC with 5% CO 2 , cells not invading to the lower chamber were wiped away. The lower surface of the transwell insert was treated with 4% paraformaldehyde for 15 min, and then stained with 0.1% crystal violet for 10 min. Finally, 5 fields were casually chosen by an inverted microscope for photographing and cell counting. The assay was repeated 3 times.
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