Polyethylene terephthalate membrane

The co-culture system was used to differentiate human SKPs into CEC-like cells. Human SKPs spheres were separated to single cells by 0.05% trypsin-0.02% EDTA, and then they were plated into 6-well plates coated with 10 mg/ml chondroitin sulphate and 10 μg/ml laminin at a density of 5 × 10⁵ cells/ml. The immortalized HCECs population B4G12 cells were cultured at a density of 1 × 10⁵ cells/ml in six-well transwell inserts with 0.4 μm diameter pores and a polyethylene terephthalate membrane (Corning). Subsequently, the Transwell inserts were added to the 6-well plates and co-cultured with the human SKPs in human endothelial-SFM supplemented with 10 ng/ml human recombinant bFGF for 8 days. The medium was changed every 2 days and Transwell inserts were changed every 4 days. Our observations confirmed that the B4G12 cells plated in the upper compartment were unable to migrate through the membrane pores into the lower compartment, whereas the secreted cytokines were free to move. Moreover, cell morphology could be clearly observed every day through the Transwell inserts. The cells were cultured at 37 °C in a 5% CO₂ humidified atmosphere. Cells were passaged when they became confluent, and cultured in human endothelial-SFM supplemented with 10 ng/ml bFGF.

Cell migration assay was carried out by transwell chamber (Corning, New York, NY) by using a polyethylene terephthalate cell culture chamber with an 8 μm pore size. Specifically, in the upper chamber, a total of 5 × 10⁴ cells were plated and cultured with serum-free medium, of which the volume in the upper chamber was 300 μl. 600 μl of 10% FBS medium was added to the lower chamber. After incubating for 24 hrs at 37℃, cells were fixed with 4% paraformaldehyde for 15 mins, stained with Crystal Violet Staining Solution for 10 mins, and washed with phosphate buffer saline (PBS). The migrated cells were observed and photographed by an inverted microscope.
Cell invasion assays were performed by using penetrating 8-μm pore size polyethylene terephthalate membrane (Corning, New York, NY). Before the invasion assays started, Matrigel was pre-coated into the upper chamber and incubated. Glioma cells were added into the upper chamber and 600 μl culture medium with 10% FBS was added into the lower chamber. After incubation, the cells were fixed with 4% paraformaldehyde for 15 mins and stained with Crystal Violet Staining Solution for 10 mins. The invasion cells were photographed by an inverted microscope.

The cell invasion assay was performed in transwell chambers. The transwell chambers were placed in a 24-well plate, and each chamber contained an insert with an 8 μm pore size polyethylene terephthalate membrane (Corning Life Sciences, MA, USA). The treated MKN-45 cells were resuspended and seeded in the upper chambers in a serum-free medium. Cells at a density of 5 × 10⁴ cells/well (in 200 μL) were seeded in the upper transwell chambers, in which the membrane was coated with Matrigel (BD Biosciences, MA, USA) and 500 μL of complete growth medium was added to the bottom chambers. The noninvaded cells in the upper chamber were removed with cotton swabs. Invaded cells on the bottom surface of the membrane were fixed, stained with crystal violet, and observed using a microscope.

Cell migration and invasion assays were performed using chambers containing a polyethylene terephthalate membrane (Corning Inc., Corning, NY, USA). Briefly, 1 × 10⁵ cells were suspended in serum-free medium and added to the upper chamber, which was coated with (invasion assay) or without (migration assay) Matrigel. Complete medium containing 20% fetal calf serum was placed in the lower chamber. After culturing for 24 h (migration assay) or 40 h (invasion assay), the migrated or invaded cells at the bottom of the membrane were fixed, stained, and counted from nine random fields under an inverted microscope (200 × magnification, Nikon Corporation, Tokyo, Japan), respectively.

THP-1 monocyte transmigration through endothelial cell monolayer was performed as described previously [37 (link)]. Briefly, hECs were grown to confluence on type I collagen-coated transwell inserts (0.9 cm² culture area, 8 μm pore size polyethylene terephthalate membrane, Corning, New York, NY, USA) before treatment with NE, variant inhibitors, or isolated neutrophils (1 × 10⁶ cells/well) for 16 h. Then, hECs were washed with blank media to remove remaining reagents or neutrophils before adding BCECF-AM-labelled THP-1 cells (1 × 10⁵) for transmigration for two hours with neutrophil-pretreated hECs and for six hours with NE-pretreated cells. THP-1 cells were labeled with BCECF-AM (Abcam, Waltham, MA, USA) at 1 nM/10⁶ THP1 cells for one hour at 37 °C and washed with PBS before being used for transmigration assay. After transmigration, cells from the apical side were removed with cotton swab, and membrane was cut, fixed, and placed in microscopic slides. THP-1 cells transmigrated through the hEC monolayer on the bottom side of the membrane were observed under an inverted phase contrast microscope (Keyence BZ-9000E, Magnification X20/0.75NA), and fluorescence images (Excitation 488 nm, Emission 520 nm) were captured. For quantification, monocytes were counted in five random microscopic fields per well, and the mean number of cells per field was calculated.

The transcellular transport study
was performed as reported previously.^{40 (link)} In brief, the cells were grown in a HTS transwell 96 well permeable
support (pore size 0.4 μm, 0.143 cm² surface area)
with a polyethylene terephthalate membrane (Corning Life Sciences,
Lowell, MA, USA) at a density of 1.125 × 10⁵ cells/well.
The cells were preincubated with M199 at 37 °C for 30 min. Subsequently,
transcellular transport was initiated by the addition of M199 to apical
compartments containing 10 μmol/L test compounds and terminated
by the removal of each assay plate after 2 h. The aliquots in the
opposite compartments were subjected to be measured for compound concentration
by LC–MS/MS. Permeability was calculated using the permeated
compound concentration.