Murine infections with Trypanosoma brucei

Six- to 8-week-old female C57BL/6J mice (JAX, stock 000664) and C57BL/6-Tg(Nr4a1-EGFP/Cre)820Khog/J strain, also known as Nur77^GFP reporter mice (JAX, stock 016617), or the Nur77^Tempo reporter mouse line (kindly provided by Dr. David Bending), were inoculated by intraperitoneal injection with approximately 2 × 10³ parasites of strain T. brucei brucei Antat 1.1E [35]. Parasitaemia was monitored by regular sampling from tail venesection and examined using phase microscopy and the rapid “matching” method [36]. Uninfected mice of the same strain, sex, and age served as uninfected controls. Mice were fed ad libitum and kept on a 12 h light–dark cycle. All the experiments were conducted between 8 h and 12 h. When using the Nur77^GFP or the Nur77^Tempo reporter mice, sample acquisition and analysis was conducted without ex vivo stimulation to preserve the TCR-dependent fluorescent reporter signal found in the tissue. For sample collection, we focussed on 30 dpi, as this has previously been shown to correlate with parasite infiltration in the epidural space [10,11]. Culture-adapted T. brucei Antat 1.1E whole cell lysates were prepared as followed. Parasites were cultured in HMI-9 supplemented with 10% FBS and 1% Penicillin/Streptomycin were grown at 37°C and 5% CO₂ and harvested during the log phase. The parasites were harvested by centrifugation (800 g for 10 min at 4°C), washed 3 times in 1× PBS (Gibco) supplemented with cOmplete protease Inhibitor cocktail (Roche), and sonicated with 5 pulses of 10 s each. The resulting lysate was cleared by centrifugation (3,000g for 10 min at 4°C to remove cell debris), and the protein concentration of the cleared supernatant was measured using the Qubit protein kit (Thermo) and kept at −80°C until usage for ELISPOT and ELISA. For LTBR-Ig treatment, mice were inoculated with 1 μg/μl of either LTBR-Ig or IgG2a i.p. (100 μl/mouse) for 4 consecutive days prior to infection, and then every 7 dpi until culling. Preparation of single-cell suspension from skull meninges for single-cell RNA sequencing.