Serum-induced chemotactic activity was investigated using 24-well Transwell chambers with inserts (Corning, Glendale, Arizona, USA). UKF-PeC-3 and UKF-PeC-4 cell lines, pre-stained with CellTracker Green CMFDA (Thermo Fisher Scientific, Darmstadt, Germany), were incubated in the absence or the presence of 20 mol/L capivasertib for 72 h. PeCa tumor cells (6 × 105/mL) were placed in the inserts (Corning, Glendale, AZ, USA) in a cell culture medium containing 10% serum. The lower chamber contained cell culture medium with 30% serum (Gibco, Thermo Fisher Scientific, Darmstadt, Germany). After incubation of 24 h at 37 °C, the lower surface and insert of the Transwell membrane were washed and gently wiped with a cotton swab to remove not migrated cells. Cells that had migrated to the lower surface of the membrane were assessed and counted by Sapphire Biomolecular Imager (Azure Biosystems, Inc., Dublin, CA, USA). Each experiment was done in triplicate.
24 well transwell chambers with inserts
Manufactured by Corning
Sourced in United States
The 24-well Transwell chambers with inserts are a versatile laboratory tool designed for various cell culture applications. The product consists of a 24-well plate and removable inserts, providing a platform for creating distinct cell culture compartments. The inserts feature a porous membrane that allows for the exchange of media, nutrients, and cellular interactions between the upper and lower chambers. This design enables researchers to study processes such as cell migration, permeability, and co-culture experiments.
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2 protocols using 24 well transwell chambers with inserts
1
Chemotactic Activity of PeCa Cells
2
Assessing Cellular Invasion and Migration
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The invasion capacity was assessed using 24-well Transwell chambers with inserts (Corning Incorporated, Corning, NY, USA). Prior to the assay, Matrigel (BD Biosciences, San Jose, CA, USA) was added to coat the membrane (8 µm pore size) in each well, and the chambers were incubated at 37 °C for 2 h. Next, the upper chambers were loaded with 5 × 104 cells resuspended in 200 µL of FBS-free culture medium. The lower chambers were covered with 500 µL of culture medium supplemented with 20% FBS. After a 24-h incubation at 37 °C with 5% CO2, the noninvasive cells were gently removed with a cotton swab. The invasive cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, washed extensively with phosphate-buffered saline, and air dried. The migration assay procedures were identical to the invasion assay procedures except that the membranes were not precoated with Matrigel. Finally, the cells that had traversed the membranes were imaged and counted under an inverted microscope (Olympus, Tokyo, Japan).
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