Polyester membrane

Endothelial cells were seeded at 50,000 cells per insert well (0.4 μm pore size polyester membrane from Corning, Inc., Corning, NY, USA) and cultured for 1 to 3 days to allow the growth of a confluent monolayer. After the different groups received different treatments, the endothelial cell monolayers were treated with 100 ng/ml LPS for 6 h before testing for permeability. Paracellular and transcellular permeability were tested as described previously [9 (link)].

Transwell coculture systems (0.4 µm pore size, polyester membrane, Corning) were used for C. albicans or G. vaginalis biofilm formation (Figure 1). In brief, C. albicans was grown overnight at 37°C in yeast peptone dextrose, harvested, washed twice with phosphate-buffered saline (PBS), and then resuspended at a concentration of 1 × 10⁶ cells/mL in RPMI supplemented with 10% FCS. Next, 500 µL of C. albicans cell suspension was seeded in triplicate on the insert membranes placed in a 24-well tissue plate and incubated at 37°C to allow the biofilm production.
G. vaginalis was grown in brain heart infusion supplemented with 2% (w/w) gelatin, 0.5% (w/w) yeast extract, and 0.1% (w/w) starch for 24 hours at 37°C with 10% CO₂ and, then, diluted to a final concentration of approximately 10⁷ colony-forming units (CFUs)/mL. Next, 500 µL of G. vaginalis suspension was seeded in triplicate on the insert membranes and incubated as described above to allow the biofilm production.
The next day, the culture medium was removed, and the inserts were washed once with PBS to eliminate nonadherent microbial cells. Biofilm formation was assessed by the crystal violet assay.

Permeability was assessed by measuring the flux of FITC-dextran from the upper compartment to the lower compartment in Transwell plates (pore size 0.4 μm; polyester membrane, Corning, New York, USA). Caco-2 cells were grown until full confluency in 12 well Transwell plates. Here, we applied the same treatment groups as described in the impedance-based technique. Cells were treated with 1000 μM H₂O₂ or pretreated with 10 μM olaparib for 30 minutes. After 24 hours, FITC-dextran solution (1 mg/ml) was added to the upper chamber. 1 hour later, a medium from the lower chamber was collected and the fluorescence intensities were detected by a Promega GloMax plate reader (Promega, USA) at 490- nm excitation and 510–570 nm emission wavelengths.

ECs were seeded in the upper chamber in 24-well culture plates (0.4 μm pore size polyester membrane from Corning, Inc.) and cultured for 2 to 3 days to produce a confluent monolayer. Then, cells were treated with LPS (100 ng/mL, Sigma) for 6 hours before permeability was tested, as previously described [23 (link)]. After adding 10 μL 40 kDa fluorescein isothiocyanate- (FITC-) Dextran (Sigma-Aldrich) to each upper insert and incubating for 40 minutes in an incubator, 100 μL medium from the upper and lower chambers was withdrawn. Then, the medium was transferred to a 96-well plate and read using excitation and emission wavelengths of 490 nm and 530 nm, respectively.

A transwell coculture system was used to investigate MSC paracrine protective effects on endothelial barrier. MSCs with or without overexpression HGF gene were seeded to upper transwell chamber (0.4 μm pore size polyester membrane from Corning, Inc., USA), and PMVECs were seeded to lower chamber (six-well culture plates). We cultured for 1-3 days to allow the growth of a confluent monolayer and then added LPS (100 ng/ml, Sigma, USA) to PMVECs.
Gram-negative bacterial pathogen lipopolysaccharide (LPS, 100 ng/ml, Sigma, USA) was treated with PMVECs to mimic endothelial barrier dysfunction. To determine the roles and mechanisms HGF, recombinant murine HGF (20 ng/ml, ProSpec, Israel) was introduced to LPS-induced PMVECs. Doses of LPS and HGF were applied according to our preliminary experiments. Moreover, PBS was applied as negative control and Akt inhibitor AZD53631 (1 μM, Selleck, USA) or PKC inhibitor enzastaurin (LY317615) (1 μM, Selleck) was applied to inhibit the activation in PMVECs. Phosphatidylinositol 3,4,5-trisphosphate as PI3K facilitates (PtdIns(3,4,5)P3, 25 ng/ml, Sigma) was also used to active mTORC2 signaling [19 (link)].