Opti memtm media

BHK21.C13 (Mesocricetus auratus-derived kidney cells; ATCC^® CCL-10) and HEK293T (ATCC^® CRL-3216) cells were maintained in Opti-MEM^TM media (Thermofisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermofisher Scientific) and incubated at 37°C in a humidified atmosphere at 5% CO₂.
Queen Saovabha Memorial Institute (Bangkok, Thailand) provided a Thai RABV strain (RABV). The virus was isolated from the brain of a rabid dog, propagated in mouse brains for seven passages, and then cultured in BHK21.C13 cells for five passages to increase virus titers.

BHK21 or hBST2-expressing BHK21 cells were seeded in 6-well plates (2.0 x 10⁵ cells/well) and infected in triplicate with RABV at an MOI of 0.01. After 1 hour of virus adsorption, cells were washed three times with phosphate-buffered saline (PBS) and maintained in Opti-MEM^TM media (Thermofisher Scientific) supplemented with 2% FBS (Thermofisher Scientific) at 37°C in a humidified atmosphere at 5% CO₂. At 0, 24, 48, 72, and 96 hours post-infection (hpi), supernatants containing RABV particles were collected and subjected to virus titration and RT-qPCR to quantify RABV infectious particles and viral RNA, respectively.
BHK21 and hBST2-expressing BHK21 cells were infected with RABV at an MOI of 1 in triplicate to examine intra- and extracellular RABV titers. Cell supernatants and pellets were collected after 8, 24, and 48 hours. Virus particles in the cell pellet fraction (in 200 μl Opti-MEM^TM) were obtained after three freeze-thaw cycles (liquid nitrogen-room temperature cycles) and supernatants were collected by centrifugation at 5,000 x g for 10 minutes. To quantify RABV infectious particles and viral RNA, the supernatants were subjected to the 50% tissue culture infectious dose (TCID50) assay and RT-qPCR, respectively.