Migration was tested with the BD Falcon FluoroBloksystem (BD Biosciences, Franklin Lakes, NJ, USA) in 24-well inserts. A total of 2.5 × 104 DU 145 or PC3 cells treated with control RNA or TRPM4 siRNA were loaded into this system in RPMI-1640 medium containing 1.0% FBS. The inserts were placed in RPMI-1640 medium with 10% FBS as an attractant. After 48 h, the cells were fixed with methanol and stained with DAPI and migrating cells were analyzed on the backside of the membrane by fluorescence. The experiment was repeated three times, and the migrated cells of at least three individual images were automatically counted using NIS-Elements AR Software (Nikon).
Falcon fluoroblok system
Manufactured by BD
Sourced in United States
The BD Falcon FluoroBlok system is a cell culture insert system designed for cell-based assays. It provides a platform for studying cell migration, invasion, and co-culture experiments using fluorescence detection.
Automatically generated - may contain errors
4 protocols using falcon fluoroblok system
1
Cell migration assay with TRPM4 knockdown
2
Transient SEC62 Depletion in CAL-120 Cells
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We compared the migratory potential of CAL-120 cells, which were transfected with control siRNA or one of two different SEC62-targeting siRNAs in the BD Falcon FluoroBlok system (BD, Franklin Lakes, NJ, USA). In addition, proliferation rates of the siRNA-treated cells were analyzed in real-time in the xCELLigence SP system (Roche Diagnostics GmbH, Mannheim, Germany). These siRNAs were previously established for various other cell types and were shown to cause efficient transient Sec62 depletion with little effect on cell proliferation during 96 h of cell growth [17 (link)]. Because of this latter observation by different laboratories, and as we wanted to be able to compare our results with those found for other tumor cells, we applied transient knock-down with siRNAs rather than CRISPR/Cas9 mediated knock-out strategies.
3
Chemotactic Response Assay Using FluoroBlok
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The chemotactic response was assessed with the BD Falcon FluoroBlok system (BD Biosciences) with pore sizes of 8.0 μm in 24-well inserts. Cells (2×104 cells) transfected with control, SDC4 or Slug siRNA were loaded into the inserts in 200 µl of DMEM medium containing 0.5% FBS in the presence or absence of TGF-β1 (5 ng/ml). Lower wells of the plate were filled with 800 µl of DMEM supplemented with 10% FBS as an attractant. After 24 h, the lower side of the membrane were fixed with methanol and mounted on glass slides for DAPI staining. DAPI-positive migrated cells were counted with a fluorescent microscope. Assays were performed in triplicate with 3 separate microscope fields per membrane.
4
Cell Migration Analysis via FluoroBlok
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Cell migration was analyzed using the BD Falcon FluoroBlok system (BD, Franklin Lakes, NJ, USA) with 8-μm pore inserts for 24-well plates. 5 × 104 UM-SCC1 cells transfected with either siRNA or plasmids were loaded into the inserts in normal medium containing 1% FBS. The inserts were then placed in the wells of a 24-well plate in medium with either 10% FBS as a chemoattractant for migration or without FBS (negative control). After 72 h, the cells were fixed with methanol, the nuclei were counterstained with DAPI and the number of migrated cells was analyzed using a bottom reading fluorescence microscope. Three representative images of each insert were obtained, and the number of migrated cells was quantified using NIS-Elements AR software version 3.2 (Nikon, Tokyo, Japan). All cell migration experiments were repeated three-fold (n = 3), and a triplicate of every cell population was analyzed in each experiment.
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