An invasion assay was performed in Matrigel-coated (BD Biosciences, Bedford, MA, USA) Transwell inserts (6.5 mm; Costar, Cambridge, UK) containing polycarbonate filters with a pore size of 8 µm. Briefly, the inserts were pre-coated with 100 µl Matrigel matrix (1 mg/ml) at 37°C for 4 h for gelling. The HTR-8/SVneo cells (1×105 cells) in 200 µl serum-free medium were plated in the upper chamber, whereas medium with 10% FBS was added to the lower well. After incubating for 24 h, the cells on the Matrigel side of the insert were scraped by a cotton swab. The inserts were then fixed in methanol for 10 min at room temperature and stained with hematoxylin and eosin (H&E; Zhongshan Goldenbridge). The cells which had invaded to the other side of the insert were counted under a light microscope (IX51; Olympus, Tokyo, Japan) in ten random fields at magnification of x200. The assay was repeated three times, and the results are represented as the percentage means of invasion ± standard deviation (SD) compared with the control.
Transwell inserts 6.5 mm
Manufactured by Corning
Sourced in United States
Transwell inserts (6.5 mm) are a laboratory product designed for cell culture experiments. They consist of a permeable membrane insert that can be placed into a larger well or dish, creating a two-compartment system for studying cell migration, transport, and other cellular processes. The core function of these inserts is to provide a controlled environment for monitoring and analyzing cellular interactions across a selectively permeable barrier.
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3 protocols using transwell inserts 6.5 mm
1
Matrigel Invasion Assay for HTR-8/SVneo Cells
2
Matrigel-coated Transwell Invasion Assay
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The invasion of cells was measured in matrigel-coated (BD, Franklin Lakes, NJ, USA) transwell inserts (6.5 mm, Costar, Manassas, VA, USA) containing polycarbonate filters with 8-μm pores. The inserts were coated with 50 μl of 1 mg/ml Matrigel matrix according to the manufacturer's recommendations. A quantity of 2 × 105 cells in 200 μl of serum-free medium were plated in the upper chamber, whereas 300 μl of medium with 10% fetal bovine serum were added to the lower well. After 24 h incubation, the cells that invaded the lower surface of the membrane were fixed and stained. Five random fields were counted at ×10 magnification for each membrane. Migration assays were similar to invasion assay except that the transwell insert was not coated with matrigel.
3
Matrigel Invasion and Motility Assay
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Invasion of cells was measured in Matrigel (BD, Franklin Lakes, NJ, USA) -coated Transwell inserts (6.5mm, Costar, Manassas, VA, USA) containing polycarbonate filters with 8-μm pores as detailed previously [21 (link)]. The inserts were coated with 50μl of 1mg/ml Matrigel matrix according to the manufacturer's recommendations. 2×105 cells in 200μl of serum-free medium were plated in the upper chamber, whereas 600μl of medium with 10% fatal bovine serum were added to lower well. After 24hrs incubation, cells that migrated to the lower surface of the membrane were fixed and stained. For each membrane, five random fields were counted at ×10 magnification. Motility assays were similar to Matrigel invasion assay except that the Transwell insert was not coated with Matrigel.
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