Precellys 24 homogeniser

Total genomic DNA (gDNA) was recovered from fungal cells by a standard nucleic acid extraction protocol adapted from [56] (link), using phenol∶chloroform and glass beads in a Precellys 24 homogeniser (PeqLab, Germany). The fingerprinting PCR reaction was performed as described previously [57] (link). Primers used were the M13 primer (GAGGGTGGCGGTTCT) and a primer consisting of the repeat sequence (GACA)₄. Products were separated by 1.2% agarose gel electrophoresis for 6 h at 3 V/cm and detected by ethidium bromide staining.

Mice were sacrificed, and the corpus callosum was dissected and stored in liquid nitrogen. Samples were homogenised in PBS containing protease/phosphatase inhibitor (Thermo Scientific) using a Precellys 24 homogeniser (Peqlab), and lysed in RIPA buffer (25 mM TRIS, 150 mM NaCl, 1 % NP-40, 0.5 % Na-deoxycholate, 0.1 % SDS, pH 7.2). Quantitative cytokine determination was performed using an electrochemiluminescence ELISA (Mouse ProInflammatory Ultra-Sensitive Kit, Meso Scale Discovery). Signals were measured on a SECTOR Imager 2400 reader (Meso Scale Discovery). For NF-kB measurements, nuclear extracts were created from whole brain homogenates (Nuclear Extraction Kit, #2900, Millipore), and NF-kB activity in nuclear extracts was determined with a commercial assay (EZ-TFA assay, #70-610, Millipore) according to the manufacturer’s instructions using a microplate reader (FLUOstar Omega, BMG Labtech). All measurements were performed in duplicates.

Approximately half of each spleen and the right-hand side lobes of the lungs from mice in the immunogenicity group were used to determine the number of viable BCG bacteria in each organ six weeks after vaccination. Tissues were removed aseptically in a microbiological safety cabinet and placed in sterile 2 ml screwcap vials containing 500 μl PBS + 0.05% Tween80 and Precellys 1.4mm ceramic beads (CK14; Peqlab, Sarisbury Green, UK). A Precellys 24 homogeniser (Peqlab) was used to homogenise tissues for 15 s at 5000 rpm before plating. Each entire homogenate was plated onto two 7H11 agar plates containing 10% OADC supplement (Yorlab, York, UK) and 0.5% glycerol. Colonies were counted after 3 weeks of incubation at 37°C.