24 transwell plate

A monolayer of Caco2 cells, cultured on semi-permeable polycarbonate surfaces, was used to study the permeability in the apical to basolateral direction. The process was atomized by a robotic Tecan EVO platform (Männerdorf, Switzerland) and 24-transwell plates from Costar (Cambridge, MA, USA). HBSS buffer pH 7.4 was dispensed to the basal side of the monolayer. The assay was initiated by adding the test substrate (10 μmol/l in HBSS buffer pH 6.5) to the apical side of the monolayer. Samples for quantitative analysis by LC/MS/MS were withdrawn before the addition of the test substrate and at 45 and 120 min post addition of the test substrate. During incubation the transwell plates were placed in a shaking incubator at 37°C between sampling. The peak areas were exported to Excel where Papp-values and recoveries were calculated. Twenty-two reference compounds with known human oral bioavailability were used to establish a correlation curve with P_app versus the fraction of the oral dose absorbed (f_a).
Efflux was studied in the same Caco2 cells with the modification that HBSS buffer pH 7.4 was used on both sides of the monolayer. The permeability was studied in the apical to basolateral direction (P_app AB) as well as in the basolateral to apical direction and (P_app BA). Efflux ratios were derived from the following equation: P_app BA/P_app AB.

The 24-transwell plates (8.0 μm pore size with poly-carbonate membrane; Corning Costar, Lowell, MA, USA) covered with 2 mg/ml basement membrane Matrigel matrix (BD Biosciences, Bedford, MA, USA) were used for the invasion assay. Each group was replicated in three separate wells. The coated transwell was hydrated with culture medium for 2 h prior to cell seeding. HMM cells were resuspended in serum-free medium and seeded at a cell density of 2.5 × 10⁴ into the upper invasion chamber. Culture medium containing 10% FBS was then added to the lower chamber. The cells were incubated at 37 °C for 24 h under normoxia or hypoxia. After a day of incubation, the cells that had invaded the lower surface of the membrane were fixed with methanol for 5 min, stained with Diff-Quik solution (Merck), and quantified by counting five random fields using a phase contrast microscope. Invasion was expressed as the ratio of invading cells incubated under hypoxia compared to the controls in normoxia.

Migration assays were performed using 8 μm polycarbonate filters in 24-transwell plates (Costar, Lowel, MA, USA). The lower sides of the filters were coated with 10 μl of type I collagen (0.5 mg/ml). CM obtained from transfected MKN1 cells mentioned above was loaded into the lower chamber and 4 × 10⁴ BAECs were seeded into the upper chamber in serum-free media and cultured for 20 h. Cells were then fixed with methanol and stained with hematoxylin (Sigma, St Louis, MO, USA) and eosin (Sigma). Cells on the upper filter surface were removed and migration was determined by counting cells that had migrated to the lower filter side under a microscope at 200×. Samples were assayed twice in triplicate.

Migration assay was performed using 24-transwell plates (Corning) with 8.0 µm pore size inserts (Corning). A total of 200,000 cancer cells were seeded in the lower chamber with complete medium. Four hours later, the complete medium was discarded and washed twice with DPBS and replaced with serum free medium. A total of 100,000 MSCs were seeded in the upper chamber with serum free medium. After 48 h, MSCs were fixed in 4% formaldehyde. Unmigrated MSCs were removed using a Q-tip and migrated MSCs that penetrated the porous membrane were stained with Hoechst 33342 (5 µg/mL), documented with a fluorescence microscope (EVOS M7000 Imaging System, Thermo Fisher Scientific) and quantified using EVOS Analysis software (Version 1.5.1479.304, Thermo Fisher Scientific).