Penicillin streptomycin solution p s

Primary human retinal pigment epithelial cells (Pr. RPE) were grown in RtEBM media (Lonza) supplemented with growth factors and 2% FBS. Human retinal pigment epithelial ARPE-19 cell line (ATCC CRL-2302), derived from normal eyes of a 19-year old male who died from head trauma in a motor vehicle accident⁶³ (link) was grown in DMEM/F12 media (Invitrogen) and Vero cells were grown in Dulbecco’s minimal essential medium (DMEM, Invitrogen) complemented with 10% Fetal bovine serum (FBS), respectively with 1% penicillin-streptomycin (P/S) solution (Invitrogen). Cells were grown at 37°C in 5% CO₂ with 95% humidity (Fisherbrand).
Zika virus strain PRVABC59 (NR-50240), initially isolated from human blood in Puerto Rico in December 2015 was acquired through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), NIH.

MDCK cells (20,000 cells/well) were seeded into 96-well plates (100 µL/well), followed by the overnight culture at 37 °C and 5% CO₂. The monolayer MDCK cells were then washed with PBS. The serum was subjected to inactivation for 30 min at 56 °C, followed by dilution with the VP medium supplemented with 1% penicillin–streptomycin (PS) solution (Thermo Fisher Scientific, USA) and 2 µg/mL L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin (Merck, Germany) to an initial dilution of 1:10, which was later diluted to twofold to a total volume of 60 µL in a 96-well plate with VP medium. The serum was first serially diluted and then mixed with an equivalent volume of virus containing 100× median tissue culture infectious dose (TCID₅₀), and the mixture solution was incubated for 1 h at 37 °C. After incubation, the serum-virus mixture solution (100 µL) was added to the monolayer MDCK cells, and the cells were cultured for 96 h at 37 °C and 5% CO₂. The titre was measured with the supernatant through hemagglutination assays. Briefly, 25 µL of 1% chicken erythrocytes were added to the culture supernatant (25 µL) and incubated for 30 min at ambient temperature, and hemagglutination was observed. Microneutralization (MN) titre was calculated as the reciprocal of the greatest serum dilution at which agglutination was inhibited.

The NC of Bester (Huso huso ​× ​Acipenser ruthenus), a hybrid species of sturgeon cultured in Bifuka Town, Hokkaido, Japan, was used in this study. Fresh NC samples were collected from the food workshop, subsequently vacuum-packed, and frozen; an uninterrupted cold chain was used to transport the material to the Faculty of Fisheries Sciences, Hokkaido University, Hokkaido, Japan. Type II collagen was purified from the NC, as described previously [21 (link)], without ethanol pretreatment. After lyophilization, the collagen was stored at −80 ​°C until use. ATDC5 cells were purchased from RIKEN Cell Bank (Tsukuba, Japan). The cell culture plates were obtained from Corning Inc. (Durham, NC, USA), and the penicillin-streptomycin (P/S) solution was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Unless otherwise specified, all other reagents used in the cell culture experiments were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA).

To verify the target relationship between miR-205/miR-181b and DES in the 3′-UTR of HSPA12B, human embryonic kidney 293 T (HEK293T) cells were maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS), 1% penicillin-streptomycin (PS) solution (Thermo Fisher Scientific) in a humidified incubator at 37°C and 5% CO₂. HEK293T cells at 60–70% confluence were transfected with HSPA12B-WT-UTR or HSPA12B-MUT-UTR together with miR-205/miR-181b (mimics). After 48 h post-transfection, the luciferase activity was measured using Dual-Luciferase® Reporter (DLR™) Assay System (Promega, Madison, WI, USA).