Establishment of minimal positive-control conditions to ensure brain safety during rapid development of emergency vaccines

Baek, Hyekyung; Kim, Kwang Ho; Park, Min Young; Kim, Kyeongryun; Ko, Bokyeong; Seo, Hyung Seok; Kim, Byoung Soo; Hahn, Tae-Wook; Yi, Sun Shin

Brain tissue collection and preparation of RNA and protein samples: Based on the EB assay results (Fig. 1) , we selected the S. enterica-derived LPS for use in the following animal experiments. The mice were divided into 8 groups (n = 4) based on mouse strain and LPS exposure time: group 1, B6/0 h; group 2, B6/2 h; group 3, B6/4 h; group 4, B6/24 h; group 5, ICR/0 h; group 6, ICR/2 h; group 7, ICR/4 h; and group 8, ICR/24 h. The LPS solution was i.p. injected into all mice simultaneously and each group of mice was sacrificed at the planned LPS PI time. After euthanasia, the brain was removed from each mouse and was cut into two hemispheres. RNA was extracted from one hemisphere by using Trizol reagent (Ambion; Life Technologies, USA) and protein was extracted from the other hemisphere by using PRO-PREP (iNtRon Biotechnology, Korea) according to the manufacturers' instructions. Briefly, for RNA extraction, the homogenized hemisphere was incubated in Trizol and chloroform reagent and then subjected to centrifugation. The aqueous phase was removed and incubated with 100% isopropanol. After centrifugation, the obtained RNA pellet was washed with 75% ice-cold ethanol and then dissolved in RNase-free water. The obtained RNA extract solution was stored at −70 o C after undergoing 60 o C heat incubation. For protein extraction, the homogenized hemisphere of each mouse was incubated in PRO-PREP solution and centrifuged several times under ice-cold conditions. The refined protein extract solution was stored at −20 o C.