24-well BioCoatTM Matrigel chamber inserts (BD Bioscience, San Jose, CA, USA) were used for this experiment. The upper surfaces of invasion chambers were coated with 30 μl of 0.5 mg/ml growth factor-reduced Matrigel (BD Bioscience, San Jose, CA, USA), and lower surfaces were coated with 20 μl of 0.5 mg/ml fibronectin (Sigma-Aldrich Korea, Seoul, Korea). Before starting the experiment, coated inserts were rehydrated with RPMI1640 for 4 hours in a humidified 5% CO2 incubator. Cells (5x104) in 500 μl of media containing 0.1% FBS were plated into invasion chambers 36 hours after SCR or ZHX1 siRNA transfection. RPMI1640 medium containing 10% FBS (700 μl) was added to each well as a chemoattractant. Mitomycin C (Sigma-Aldrich Korea, Seoul, Korea) was used to inhibit cholangiocarcinoma cell proliferation. After incubation for 18 or 80 hours, non-invading cells on upper insert surfaces were removed cleaned by scraping. Invaded cells in inserts were fixed and stained using Diff-quik solution (Sysmex, Kobe, Japan). Experiments were performed in triplicate. Pictures were taken under a light microscope at 20x and 56x. Total numbers of invaded cells were counted using Adobe Photoshop CS6 software.
Biocoat matrigel chamber inserts
Manufactured by BD
Sourced in United States
BioCoatTM Matrigel chamber inserts are a laboratory product used for cell culture and invasion assays. The inserts are pre-coated with a basement membrane extract, providing a physiologically relevant extracellular matrix for studying cell migration and invasion.
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2 protocols using biocoat matrigel chamber inserts
1
Invasion Assay Using Matrigel Chambers
2
Measuring FBS-Induced Cell Invasion
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To examine FBS-induced cell invasion, we used 24-well BioCoatTM Matrigel chamber inserts (BD Bioscience, San Jose, CA, USA). Upper surfaces of inserts were covered with 30 µL of 0.5 mg/mL of growth factor-reduced Matrigel (BD Bioscience), and lower surfaces of inserts were covered with 20 µL of 0.5 mg/mL fibronectin (Sigma-Aldrich). Coated inserts were first rehydrated with culture medium for 4 h in a CO 2 incubator. Within 1-2 days after SCR or ZHX1 siRNA transfection and immediately after preparing Mock or ZHX1-over cells, 5 × 10 4 cells in 500 µL of media containing 0.1% FBS were added to inserts. To observe chemotactic cell migration, 700 µL of culture medium containing 10% FBS was added to each well. Mitomycin C (100 or 300 ng/mL; Sigma-Aldrich) was used to inhibit glioblastoma cell proliferation. After incubation for 70 or 90 h, cells on upper surfaces of inserts were removed by scraping. Cells that had penetrated inserts were fixed and stained using Diff-Quik solution (Sysmex). Total numbers of invasive cells were counted using Adobe Photoshop CS6 software. Experiments were performed in triplicate.
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