Speedmill plus homogenizator

Tumors were collected and digested in Invitrogen™ ProcartaPlex™ cell lysis buffer (ThermoFischer Scientific, Austria) using SpeedMill Plus homogenizator (AnalytikJena, Germany). Tumor lysates were stored at −80°C until use. Cytokines were determined using LEGENDPlex™ mouse inflammation panel (BioLegend, Germany) according to the manufacture's protocol. IL‐28 was measured by IL‐28 ELISA kit from PBL Assay Science (Piscataway, NJ) according to the manufacture's protocol.

Total RNA was isolated from mock-, IFN-γ or IL-4-stimulated BMDMs generated from CD83wt or CD83 cKO mice. Cell pellets were lysed in RLT+β-Mercaptoethanol extracted by RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. In addition, wound biopsies were collected from CD83wt as well as cKO mice at day 0, day 3 or day 6, stored in RNAlater (Qiagen) at -80°C and subsequently used for further analyses. Homogenization of the tissue was performed in RLT+β mercaptoethanol using innuSPEED lysis Tube W (Analytic Jena). We performed three homogenization cycles à four minutes in the SpeedMill PLUS homogenizator (Analytic Jena).

Individual and pooled ticks with up to 5 individuals were subjected to homogenization using the SpeedMill PLUS homogenizator (Analytikjena, Jena, Germany) and subsequently, total RNA extraction via BlackPREP tick DNA/RNA kit (Analytikjena, Jena, Germany) according to the manufacturer’s instructions. Tick pools with more than 5 individuals were homogenized by vortexing with tungsten carbide or stainless steel beads (Qiagen, Hilden, Germany) in 500–700 μl of Eagle’s minimal essential medium, supplemented with 5% foetal bovine serum and 1% L-glutamine. They were clarified by centrifugation at 1430× g for 4 min, aliquoted and stored at -80 °C. Nucleic acids purification was carried out in one aliquot via High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Mannheim, Germany). All specimens were subsequently subjected to complementary DNA synthesis with random hexamers, using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Hennigsdorf, Germany), performed according to the manufacturers’ guidelines.