Following transfection with LKB1 siRNA for 24 h, RA FLS were centrifuged and loaded onto Transwell filters with 8 mm pores (Corning, Inc., Corning, NY, USA) positioned on top of migration chambers for 24 h. DMEM containing 10% FBS was placed in the bottom chamber of the Transwell plate as a chemoattractant. Non-migrating cells on the top membrane surface were removed by washing with PBS and cotton swabs. Migrated cells were stained with crystal violet dye and counted in five random fields per sample under an inverted microscope (Olympus, Tokyo, Japan; magnification, 100×; 0.55 numerical aperture dry objective; Scale bar, 100 mm).
Transwell filters with 8 mm pores
Manufactured by Corning
Sourced in United States
Transwell filters with 8 mm pores are a type of lab equipment used for cell culture applications. They consist of a permeable membrane insert that fits into a well, allowing for the separation of cell populations or the study of cell-cell interactions. The 8 mm pore size is a common specification that enables the passage of cells and molecules while retaining larger cellular components.
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Lab products found in correlation
2 protocols using transwell filters with 8 mm pores
1
LKB1 siRNA Transfection Impacts RA FLS Migration
2
Transwell Assay for Cell Migration
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Cell migration was measured in Transwell filters with 8-mm pores (Corning, Tewksbury, Massachusetts, USA). Inserts were coated with human fibronectin protein (Cat. #33016015, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Sorted cells (2×10 4 cells), in 200 μL of medium, were loaded into the upper chambers, and 500 μL of the hOPC medium was added to the chamber. After incubation for 18 h at 37°C, a cotton swab was used to wipe the cells that had not migrated onto the upper surface of the chamber. The migrated cells were fixed with 4% paraformaldehyde and stained with DAPI (1:20). Images were acquired using a fluorescence microscope.
The migrated cells were quantified using ImageJ software [26] by analysing cell nuclei from at least five randomly selected fields per chamber. The cell migration rate was calculated as follows: cell migration rate (V1%) = N1/N2 × 100%, where N1 refers to the initial cell seeding number, and N2 refers to the number of migrated cells. The experiment was repeated three times independently in the laboratory.
The migrated cells were quantified using ImageJ software [26] by analysing cell nuclei from at least five randomly selected fields per chamber. The cell migration rate was calculated as follows: cell migration rate (V1%) = N1/N2 × 100%, where N1 refers to the initial cell seeding number, and N2 refers to the number of migrated cells. The experiment was repeated three times independently in the laboratory.
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