Millicell hanging cell culture insert

The migration of UM cells was evaluated with a wound healing scratch assay. Cells were seeded in 6-well plates (5 × 10⁵/well) until reached full confluence. Cells were serum-starved overnight and an artificial scratch wound was created at the center of the well and photographed, the scratch areas were photographed at 0, 24 and 48 h. Migration index was calculated as the follows: [(original scratch width-scratch width at 24/48 h)/(original scratch width)] × 100%. The assay was repeated three times.
Transwell invasion assays were performed to assess cell invasion. 24-well Millicell hanging cell culture inserts (8.0 µm, Millipore, USA) and Matrigel (BD Biosciences, USA) were used as per manufacturer’s protocol. Approximately 2 × 10⁵ cells in serum-free medium (100 ul, 2 × 10⁶ cells/ml) were added per well to the upper chamber, 500 ul complete medium with 10% FBS in the lower chamber served as a chemoattractant. After 48 h of incubation, cells that had invaded through the Matrigel were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet for counting. Three separate fields were counted for each filter with microscope (UOP-DSZ2000X; Chongqing, China). The assay was also repeated three times.

hCMEC/D3 cells were seeded on Millicell Hanging Cell Culture Inserts (Millipore Corp, Billerica, MA, United States) at a density of 2 × 10⁵ cells per well. Following the formation of monolayer confluence, the cells were treated with a medium containing 20 μg/ml arginase for 24 h. Fluorescein (10 μg/ml) and FITC-dextran (3 kDa) (1 mg/ml) were added to luminal chamber. Concentrations of FITC-dextran and fluorescein in the receiver at designed times (1, 1.5, 2 h) were measured on SpectraMax Gemini XPS microplate fluorometer (Sunnyvale, CA, United States) at 430 nm (excitation) and 525 nm (emission) for fluorescein and 485 nm (excitation) and 525 nm (emission) for FITC-dextran. Apparent permeability coefficients (P_eff) were calculated as previously described (Förster et al., 2008 (link)).

The assays were performed using Millicell™ hanging cell culture inserts (polyethylene terephthalate (PET) membranes with 8 μm pores) (Millipore). A total of 4 × 104 cells were plated in medium containing 2% serum in the upper chamber on a 10% Matrigel-coated membrane, while the lower chamber was filled with medium containing 2% serum unless stated otherwise. After incubation for 72 h, the cells in the upper chamber were removed and the invaded cells at the bottom of the membrane were fixed with 4% Paraformaldehyde (PFA). The invaded cells were then treated with methanol and stained with 0.5% crystal violet. To quantify the invaded cells, the crystal violet stained cells were washed with water and then lysed with 10% acetic acid. The absorbance of the lysates was measured at 595 nm. The OD values of the invaded cells are shown in the graphs. The quantification was determined from three independent experiments.

Cell migration and invasion were evaluated using a 24‐well Millicell^® hanging cell culture inserts (8.0 μm pore size; PIEP12R48) purchased from Millipore Corporation (Billerica, MA, USA) with or without Matrigel (B&D Biosciences, San Jose, CA, USA). Cells (5 × 10⁴) starved overnight were added to the upper chamber in serum‐free medium, and the bottom chamber was filled with conditioned medium containing 10% FBS. After 24 hrs of incubation, the cells in the upper chamber were wiped and removed with a cotton swab. Cells that migrated or invaded to the other side of the chamber were fixed with 100% ethanol and then stained for 30 min. with 0.1% crystal violet. The cells were then counted under a microscope at 100× magnification. Five fields from each sample were randomly selected, and the average number of cells in each field was counted and presented as mean ± S.D.

The invasion assay was performed using Millicell™ hanging cell culture inserts (polyethylene terephthalate (PET) membranes with 8 μm pores) (Millipore). HMEC-1 cells (1 × 10⁵ cells per well) were plated on the upper chamber and allowed to grow to confluence, and then 10% Matrigel was loaded into the chamber. Tumor cells were treated with 50 ng/ml EGF in serum-free medium and then stained with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) (Invitrogen) for 30 min. DiI-stained tumor cells (2 × 10⁵) were then loaded into the chamber, which was filled with serum-free medium, and incubated for 2 days. Cells on the apical side of each insert were scraped off. Invasion to the basolateral side of the membrane was visualized using an immunofluorescent microscope. The number of invading cells was determined in three randomly chosen fields under the microscope for three independent experiments.

Cells were seeded on Millicell Hanging Cell Culture Inserts (Millipore, Billerica, MA, USA) for TER determination using an epithelial voltohmmeter Millicell ERS-2 (Merck Millipore, Billerica, MA, USA).