Rpmi 1640 medium

The three different interventions were as follows: RPMI-1640 medium (Gibco, USA) continually supplemented with 2 g/L glucose and 10% FBS as a normal control (CON) diet for 24 h cycles; Normal control RPMI-1640 medium for 6 h followed with serum-free and glucose-free RPMI-1640 medium for 18 h for 24 h cycles as a TRF mimicking group; 0.5 g/L glucose and 1% FBS were continuously added to glucose-free RPMI-1640 medium as a simulated fasting condition (STS) for 24 h cycles. When changing the medium, cells were washed twice with PBS and then switched to another medium.

The American Type Culture Collection (Manassas, VA, USA) provided us with cell lines including HRCE, 786-O, OS-RC-2, Ketr-3 and A498. RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to culture these cell lines and environmental conditions were set at 37˚C and 5% CO₂. Additionally, 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) were added to RPMI-1640 medium for cell growth.

Between 2012 and 2019, a total of 225 dead bats of 21 bat species from 59 (out of 212) municipalities in Slovenia were collected by bat biologists or volunteers and stored frozen (S1 Table). The collected dead bats were submitted to the National Veterinary Institute, Institute of Microbiology and Parasitology, Virology unit, for lyssavirus diagnosis. Bat biologists provided information on bat species, location (municipality), and year of collection. Bat species were identified using morphological keyes, described in Dietz and Halversen [35 ]. Additional molecular characterization of the host species from lyssavirus positive bat sample PP-0868/2014, where NGS reads were mapped to cytochrome b and cytochrome c oxidase subunit I [36 (link)], confirmed the original assignment classification to Myotis capaccinii.
Brain samples were collected through the foramen occipitale magnum by pipette aspiration. After aspiration of the brain tissue, the cranial cavity was rinsed with RPMI-1640 medium (Thermo Fisher, USA). The collected brain tissue was homogenized in a total volume of 500 μl of RPMI-1640 medium (Thermo Fisher, USA) before long-term storage at < - 60°C.
Permit for capture, disturbance, and temporary taking from the wild and sampling of protected animals was issued by the Agency of the Republic of Slovenia for the Environment No. 35601-35/2010-6.

Human HT1080 cells were purchased from the American Type Culture Collection. HT1080 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Thermo Fisher Scientific). RS4;11 cells were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ) and cultured in α-MEM with ribo- and deoxyribonucleosides (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS (Thermo Fisher Scientific), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Thermo Fisher Scientific). Jurkat T cells were provided by A. Rösen-Wolff and cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS (Thermo Fisher Scientific), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Thermo Fisher Scientific). Murine thymocytes were isolated as described below and cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS (Thermo Fisher Scientific), penicillin (100 U/ml), and streptomycin (100 μg/ml) (Thermo Fisher Scientific). All cells were cultured in a humidified 5% CO₂ atmosphere.