A matrix degradation assay was performed using the Cell Invasion Assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. In brief, cells were seeded on the upper surface of the insert membrane pre-coated with matrix proteins. Degradation of the matrix proteins was necessary for seeded cells to pass through the pores of the membrane. Finally, the invaded cells were stained and quantified. For mouse primary pulmonary artery endothelial cells, a suspension of 2×10 5 cells in various concentrations of recombinant human APN/CD13 was seeded on the upper surface of the insert membrane pre-coated with basement membrane matrix solution. For EHMES-10 and MSTO-211H cells, a suspension of 2×10 5 cells containing MT95-4 (20 μg•mL -1 ) or human control IgG was seeded on the upper surface of the insert membrane pre-coated with matrix proteins. Invasive cells were counted in three random microscopic fields per well after 48 h.
Cell invasion assay kit
Manufactured by Cell Biolabs
Sourced in United States
The Cell Invasion Assay kit is a laboratory tool designed to measure the ability of cells to invade through a simulated extracellular matrix barrier. The kit provides a standardized and quantitative method to assess this cellular behavior.
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9 protocols using cell invasion assay kit
1
Matrix Degradation Assay for Cell Invasion
2
S100A2 Knockdown Impacts Wound Healing and Invasion
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HEp-2 cells were trypsinized and counted. 50 nM of S100A2-targeted siRNAs or control siRNA was transfected into HEp-2 cells (105 cells/well). After 48 hrs, wound healing and invasion experiments were performed on 6-well plates seeded with HEp-2 cells as described previously [14 (link)]. After the cells reached confluence, a 200μl pipette tip was used to incise the cell monolayer. The debris was then rinsed away and removed. The extent of gap closure was monitored and photographed under a microscope for up to 24 hours. The invasion assays were performed using a Cell Invasion Assay Kit (Cell Biolabs) according to the manufacturer’s instructions. After 24 hours, the number of cells that invaded and attached to the bottom of chamber was measured by CyQuant GR fluorescent dye (560 nm).
3
Invasion Assay of HTR-8/SVneo Cells
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Invasion assays were performed using a Cell Invasion Assay kit (Cell Biolabs, Inc., Beijing, China), according to the manufacturer's protocol. The HTR-8/SVneo (50,000) cells were resuspended in medium with PIF or PIFscr (100 ng/ml). As determined using trypan blue exclusion under a light micros'cope (CK40; Olympus Corporation, Tokyo, Japan), ~1×105 viable HTR-8/SVneo cells were seeded into the upper chamber of a 24-well plate with polycarbonate membrane inserts. The numbers of cells to invade through the ECM Matrix gel were determined using CyQuant GR fluorescent dye (560 nm).
4
Quantifying Cell Invasion Dynamics
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Cell invasion assays were performed using a Cell Invasion Assay kit (Cell Biolabs Inc.; San Diego, CA, USA) in accordance with the manufacturer's instructions. Briefly, MDA-MB-231 cells expressing shNT or shGPR81 RNA were suspended in serum-free media. The cells (1.5 × 105/chamber) were placed in inserts designed for a 24-well plate. Each insert contained a thin layer of extracellular matrix over a polycarbonate membrane (8 µm pore size). Lower compartments were filled with DMEM containing 10% FBS. After incubation for 6 h at 37 °C in a 5% CO2 incubator, cells that invaded and migrated through the matrix-containing membrane and reached the lower surface of the invasion chamber were observed using a phase-contrast microscope with an attached camera. The cells that had migrated were counted using photographic images.
5
Studying RLIP76 Protein Function
6
Comprehensive Protein Expression Analysis
7
Cell Migration and Invasion Assay
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For the cell migration and invasion assays, a Cell Invasion Assay kit (Cell Biolabs, Inc., San Diego, CA, USA) was used according to the manufacturer's instructions. Briefly, a total of ~1×104 cells were seeded into the Transwell insert in serum-free DMEM, whereas DMEM with 10% FBS was added to the lower well. At 48 h, cells were fixed and cells at the top of chamber were removed. Cells on the lower side of the chamber were stained with crystal violet, and visualized with a light microscope. The stain was dissolved with 33% acetic acid and absorbance of each well was measured at 570 nm with a microplate reader (Thermo Fisher Scientific, Inc.). The procedure was repeated independently three times, with triplicate chambers for each group.
8
Regulation of HCC Cell Proliferation and Invasion
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HepG2 cells and HuH-7 cells, from the Health Science Research Resources Bank (Osaka, Japan), were distributed onto 6-well plates (Asahi Techno Glass, Tokyo, Japan) at a concentration of 1.5 × 104 cells per 3 ml per well in Dulbecco's modified Eagle medium (Sigma-Aldrich, St. Louis, MO, USA). After overnight incubation, Pin1-specific small interfering RNA (siRNA) or negative control siRNA was transfected into HCC cells using HiPerFect Transfection Reagent (QIAGEN Inc., Valencia, CA, USA). These cells were further treated with 10 ng ml−1 tumour necrosis factor-α (TNFα) for 30 min to activate NF-κB. In some experiments, cells were incubated for 24, 48 or 72 h in medium containing 0 (vehicle only), 5, 10, 25 or 50 μmol l−1 juglone or PiB (Sigma-Aldrich). Cell proliferation was evaluated at 48 h after treatment by DNA incorporation of 5-bromo-2'-deoxyuridine (BrdU) using the Biotrak cell proliferation ELISA system (GE Healthcare, Buckingham, UK). For cell cycle analysis, cells were harvested and suspended with PBS containing 100 μg ml−1 propidium ionide and 10 μg ml−1 RNase, and analysed the content of DNA using BD FACS CANTO II (BD Biosciences, Franklin Lakes, NJ, USA). Cell invasiveness was evaluated at 48 h after treatment using the Cell Invasion Assay Kit (Cell Biolabs Inc.). Cell lysates and nuclear extracts were prepared for western blotting or EMSA.
9
Molecular Mechanisms in Cancer Cell Signaling
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