3 3 5 5 tetramethylbenzidine tmb substrate

For the in vitro permeability assay, cells (2×10⁵) were grown on a transwell insert (0.4 µm; Corning B.V. Life Sciences, The Netherlands) until a monolayer was formed. The upper chambers were reconstituted with 10% FBS-containing medium with MIF and inhibitors. At the indicated time points, the media in the upper chambers were replaced with 300 µl of serum-free media containing 4.5 µl of streptavidin-horseradish peroxidase (HRP) (R&D Systems, Inc., Minneapolis, MN). The media (20 µl) in the lower chambers were collected 5 min after adding streptavidin-HRP and assayed for HRP activity by adding 100 µl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (R&D Systems). Color development was detected using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.

HUVECs (1× 10⁵) were grown on the upper chambers of Transwell plates (0.4 μm; Corning, The Netherlands) until a monolayer was formed. The HUVECs monolayer was coincubated with BSA or NS1-treated washed platelets (1×10⁷, 200 μl) along with Tyrode’s buffer. After the indicated time point, the upper chambers were reconstituted with 300 μl serum-free media, which contained 3 μl of streptavidin-horseradish peroxidase (HRP) (R&D Systems). Fifty microliters of medium in the lower chamber was collected 10 min after adding streptavidin-HRP and was assayed for HRP activity by adding 100 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (R&D Systems). The color development was detected by a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.

Patients' clinical blood parameters were tested using the standard protocols in the Department of Pathology, NCKUH. Total circulating white blood cell counts (WBCs), platelet counts, and hematocrit were obtained by the standard complete blood count test. In addition, a DENV diagnosis was confirmed using one or more examinations: positive for plasma nonstructural protein 1 (NS1) antigen, dengue IgM antibodies detected using a kit (Bioline Dengue Duo™; Standard Diagnostics, Seoul, Korea), or DENV RNA detected using real-time reverse transcriptase-polymerase chain reaction (RT-PCR) (TIB Molbiol, Lightmix kit; Roche Applied Science, Berlin, Germany) [26 (link)]. For viral NS1 measurement, NS1 ELISA was performed using paired anti-NS1 antibodies that were prepared in our laboratory, and the ELISA was quantified by the addition of 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (R&D Systems) [27 (link)].