Macrosep advance centrifugal device

The Pall Corporation Macrosep Advance Centrifugal Devices (10 kDa, 3 kDa, and 1 kDa molecular weight cutoffs) were used to separate metabolite exudate components by molecular weight. Fractionation was performed following manufacturer instructions at 4 °C. Fractions of <1 kDa, 1–3 kDa, 3–10 kDa, and >10 kDa were collected, dried down, and analyzed in DDM or MIC assays.

IgG was prepared from pooled sera from de-identified peanut-allergic patients that had undergone OIT. IgG was purified over Nab protein G spin columns (Thermo Scientific), concentrated, and dialyzed with Macrosep Advance Centrifugal Devices carrying a 50 kDa cutoff (Pall Corporation) and filter-sterilized with 0.2 µM syringe filters (Millex). Allergen-specific IgG concentrations were determined by Phadia ImmunoCAP (ThermoFisher).

Human hepatoma cell line HuH-7, Baby hamster kidney cell line BHK-21 and Aedes albopictus cell line C6/36, purchased from the Japanese Collection of Research Bioresources (Japan) and the American Type Culture Collection (ATCC, Manassas, Virginia), were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone, Logan, UT). Human umbilical vein endothelial cells (HUVECs) were purchased from the Bioresource Collection and Research Center (BCRC) of Taiwan and maintained in endothelial Basal Medium-2 (EMB-2, Lonza, Walkersville, MD) supplemented with 10% FBS, and SingleQuots™ Kit (Lonza, Walkersville, MD). All cells were cultured at 37 °C in a 5% CO2 atmosphere, except for C6/36 which were cultured at 28 °C. Four serotypes of dengue viruses, DENV 1 (local Taiwan strain 8700828), DENV 2 (16681 and local Taiwan strain 454009 A), DENV 3 (local Taiwan strain 8700829), and DENV 4 (local Taiwan strain 59201818) were propagated in C6/36 cells as previously described^{52 (link)}. To prepare high titers of DENV, cell-free supernatants were concentrated by Macrosep® Advance Centrifugal Devices (molecular weight cutoff of 30 kDa; Pall Corp., Port Washington, NY) at 6000 × g at 4 °C and stored below −70 °C until use.

DENV serotype 2 local Taiwan strain 454009 A and ZIKV Asian strain PRVABC were propagated in C6/36 cells as previously described [51 (link)]. To prepare high titers of DENV, we concentrated DENV-containing culture supernatants with Macrosep Advance Centrifugal Devices (molecular weight cutoff of 30 kDa; Pall Corp., Port Washington, NY, USA) at 6000×g at 4°C and stored below −70°C until use. UV-inactivated DENV (UV-DENV) was obtained by treatment with UV 100 mJ/cm² for 100 s in a UV-crosslinker (VILBER, France). To deplete NS1, we incubated DENV-containing culture supernatant with mouse anti-dengue NS1 1D33-agarose (Leadgene) at 4°C for 1 h, followed by centrifugation at 3000×g at 4°C to obtain NS1-depleted DENV supernatant. The virus titer and NS1 concentration in each condition were analyzed by fluorescent focus assay and NS1 ELISA after the process.

The CEs were further purified by spin column chromatography (Pall Corporation Macrosep Advance Centrifugal Device, New York, NY, USA) as described by Chelliah et al. [8 (link)], in a batch system. The cell free supernatant (CFS) was filtered in the <30 kDa spin column, and centrifuged at 3000 rpm for 30 min. The <30 kDa filtrates were transmitted to a <10 kDa spin column, and centrifuged at 4000 rpm for 30 min. Finally, the filtrate was collected and tested for coagulation and antimicrobial activity by applying the disc diffusion method, through which partially purified low-molecular-weight peptides were tested against Escherichia coli 0157:H7, Salmonella paratyphi B, Salmonella typhimurium, Shigella flexneri, Salmonella paratyphi A, and Kelbsiella pneumoniae. The molecular weight of coagulation and antimicrobial peptide falls under 7.5–6 kDa (Figure 1b).

We used the following materials and equipment: fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), ExoQuick-TC reagent (SBI, Los Angeles, CA, USA), phosphate-buffered saline (PBS; Biosesang, Gyeonggi-do, Korea), gold(III) chloride trihydrate (HAuCl₄; Sigma-Aldrich, St. Louis, MO, USA), deionized sterile water (DW; Bioneer, Daejeon, Korea), Macrosep Advance Centrifugal Device (30 kDa; Pall Corporation, Washington, NY, USA), Micro BCA^TM protein assay kit (Thermo Scientific, Waltham, MA, USA), Dulbecco’s modified eagle medium/high glucose (DMEM; HyClone, Logan, UT, USA), Roswell park MEMorial institute 1640 medium (RPMI; HyClone, Logan, UT, USA), Minimum essential media (MEM; Welgene, Gyeongsangbuk-do, Korea), penicillin-streptomycin (Gibco, Brooklyn, NY, USA), Dulbecco’s phosphate buffered saline(DPBS; Sigma-Aldrich, St. Louis, MO, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Invitrogen, Carlsbad, CA, USA), and 5-dodecanoylaminofluorescein (DAF; Invitrogen, Carlsbad, CA, USA).