Migration assays were used to assess the recognition of canine CXCL8 by the huCXCR2 expressed on the dual CAR T cells. Migration was assessed using 8 µm transwell plates (Corning), with the lower chamber containing 25 ng/ml recombinant canine CXCL8 (R&D Systems) in serum free DMEM. Migration controls included negative controls (medium only) and positive controls (DMEM with 10% FBS). Prior to addition to the top chamber the CAR T cells were washed twice. Plates were incubated at 37˚ C for 4 hours and then the cells in the bottom well were collected and enumerated by hemocytometer.
8 m transwell plate
Manufactured by Corning
Sourced in United States
The 8-µm Transwell plate is a laboratory equipment product designed for cell-based assays. It features a permeable membrane with 8-micron pores that allows for the study of cell migration, invasion, and co-culture experiments.
Automatically generated - may contain errors
5 protocols using 8 m transwell plate
1
Canine CXCL8-driven Migration Assay for CAR T Cells
2
Investigating TALL-104 Cell Adhesion and HMVEC Migration
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TALL-104 cells were added to plates of confluent HMVECs (1:1). Co-cultures were then treated with YKL-40 (200 ng/mL) and/or a CCR5 inhibitor TAK-452 (30 nM). After 8 h, the cell medium was gently aspirated, and the cells were gently washed with PBS three times. TALL-104 cells adhering to HMVECs were imaged and quantified.
For cell migration, HMVECs (6 × 104) were loaded to the top chamber of 8 µm transwell plates (Corning, Hartford, CT, USA) and incubated for 6 h at 37 °C. After washing the insert membrane, the migrated cells were fixed, stained, and quantified.
For cell migration, HMVECs (6 × 104) were loaded to the top chamber of 8 µm transwell plates (Corning, Hartford, CT, USA) and incubated for 6 h at 37 °C. After washing the insert membrane, the migrated cells were fixed, stained, and quantified.
3
Cell Invasion Assay using Transwell
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A Transwell invasion assay was used to analyze the invasive rate of cells. Briefly, AMC-HN-8 cells in 100 µl (2×105) serum-free medium (Thermo Fisher Scientific, Inc.) were plated into the upper chambers of an 8-µm Transwell plate (Corning, Inc.) precoated with Matrigel™ (BD Biosciences) for 24 h at 37°C after transfection. RPMI-1640 medium containing 20% FBS was plated in the lower chamber to serve as a chemoattractant. Following 24-h incubation at 37°C, the invading cells on the bottom surface of the filter were fixed with 100% methanol at 4°C for 30 min and stained with hematoxylin at room temperature for 20 min. Cell invasion was analyzed in three randomly selected fields under a fluorescence microscope (magnification, ×20).
4
Transwell Invasion Assay for Cell Migration
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A Transwell invasion assay was used to analyze the invasive rate of cells. Briefly, AMC-HN-8 cells in 100 µl (2×105) serum-free medium (Thermo Fisher Scientific, Inc.) were plated into the upper chambers of an 8-µm Transwell plate (Corning, Inc.) precoated with Matrigel (BD Biosciences) at 24 h (37°C) after transfection. DMEM/F12 (Hyclone; Cytiva) containing 20% FBS was plated in the lower chamber to serve as a chemoattractant. Following the incubation (37°C, 24 h), the invading cells on the bottom surface of the filter were fixed with methanol (100%, 4°C) for 30 min and stained with hematoxylin at room temperature for 20 min. Cell invasion was analyzed in three randomly selected fields under a fluorescent microscope (magnification, ×20).
5
Transwell Invasion Assay for KYSE-150 Cells
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KYSE-150 cells were cultured in 24-well plates and transfected with specific siMPP7 or control siRNA. Forty-eight hours post transfection, cells were harvested, and single-cell suspensions in serum-free DMEM were prepared, of which 150 μL (3 × 104 cells) was seeded into the upper chambers of an 8 µm Transwell plate (Corning Life Sciences, Corning, NY, USA). The lower chamber was filled with 600 μL of DMEM supplemented with 10% FBS. For the invasion assay, the membrane was coated with Matrigel (Beyotime, Nantong, China) 6 h prior to seeding. Following incubation at 37 °C for 24 h, cells were fixed with ice-cold methanol for 20 min and stained with 0.1% crystal violet for 5 min at room temperature.
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