Rna extraction reagent kit

Primary cells were isolated from bone marrow of BALBc/c mice as described (29 (link)). To generate bone marrow derived macrophage (BMDM), the isolated cells (1×10⁶/well) were cultured in a 24-well plate for 7 days in RPMI 1640 (10% FBS, penicillin, streptomycin) containing 20 ng/ml murine macrophage-stimulating factor (M-CSF, PeproTech). On day 8, 200 µl of mice serum was diluted at 1:25 in RPMI 1640 and were incubated with the equal volume of diluted virus stocks (MOI=0.1) for 1 h at 37°C in 5% CO₂, the virus-only well as mock control. After removing the supernatant, BMDM were incubated with the serum/virus mixture for another 1 h at 37°C. 400 µl of the RPMI 1640 (4% inactivated FBS, penicillin, streptomycin) was added to each well, and then the plates were allowed to maintain for 24 h at 37°C in 5% CO₂. On the next day, the cells were collected, and total RNA was extracted with a RNA extraction reagent kit (Magen). The RNA was stored in -20°C to be analyzed.

The LD₅₀ was calculated using the Reed & Muench method. To evaluate the protective effect of zEDIII-rHF nanoparticles against ZIKV, 14 days after the final immunization, each mouse was infected with 20 µl of ZIKV (10× LD₅₀ dose, SZ-WIV01) by foot pad injection. Over the next 21 days, the mice were monitored for body weight loss and survival. Body weight loss of more than 20% was considered indicative of lethality. Mice were euthanized according to the study guidelines. The viral challenge experiments were carried out in an ABSL-2 facility. Blood was collected from all mice for viremia analysis on day 3 and day 7 postinfection. The brain, testis, kidneys, spleen, and liver were collected for viral titer detection. Serum and tissue samples were obtained from mice, and total RNA was extracted with an RNA extraction reagent kit (Magen Cat: R4173-03). A universal pair of primers (forward primer: 5’-AGGATCATAGGTGATGAAGAAAAGT-3’ and reverse primer: 5’-CCTGACAACACTAAGATTGGTGC-3’) was used to amplify ZIKV (33 (link)). All qRT-PCR assays were performed with the One-step TB Green PrimeScript™ PLUS RT-PCR Kit (TaKaRa, Code No. RR096A) on a CFX96 Touch real-time PCR detection system (Bio-Rad).