Metal bead

Tumors were established as described above. After randomization of mice (n = 4–6), they received at day 0 a single intratumoral dose of 1 × 10⁷ PFU. Mice were sacrificed at day 4 and tumors were harvested, washed with PBS, and the fluorescence signal from tumors was acquired using a GelDoc imaging system (Bio-Rad, Hercules, CA, USA) and quantified using ImageJ.
For determining viral titer and IFN-β concentrations within tumors, mice were treated as described above and sacrificed at day 4 after viral administration. Tumors were harvested, weighed, and homogenized using metal beads and a tissue homogenizer (QIAGEN, Hilden, Germany). Virus titers were determined by plaque assay on MA104 cells, and IFN-β was quantified using a mouse IFN-β ELISA kit (R&D Systems, Minneapolis, MN, USA).

Eggs were collected on juice agar plates in 30 min intervals and immediately exposed to one session of heat shock at 37°C for 30 min or kept as controls. This selection was random. Embryos were thereafter kept in a climate‐controlled 22°C incubator for approximately 2 h, dechorionated in 3.5% bleach, and staged under SMZ 745 (Nikon) bright‐field microscope using the criteria for Bownes' stage 5 (Bownes, 1975 ), including formed cells at egg surface and round pole cells at the posterior axis. Single embryos were collected in 2 μl RNase‐free water with an RNase inhibitor and ruptured with an RNase‐free needle. One 5 mm ∅ metal bead (Qiagen) and 500 μl Qiazol (Qiagen) were added per sample and the samples were homogenized for 2 min at 40 Hz using TissueLyser LT (Qiagen). n = 24 single embryos per condition.

After samples were collected for histology, RNA and protein analysis as described above, the remaining organ tissue was washed extensively in PBS and then placed into a previously weighed 2 ml Eppendorf tube containing a metal bead (Qiagen) and weighed again. 1 ml PBS was added to each tube and tissues were homogenized by bead beating in a Mixer Mill MM 400 (Retsch) for 6 min at 30 Hz at room temperature. Homogenized tissues were then 1:10 serially diluted in PBS and in triplicates plated onto agar plates. For WT ST LB agar plates containing 100 μg/ml streptomycin (plus 50 μg/mL carbenicillin for ST lux-invF and 50 μg/mL kanamycin for ST prgH-GFP respectively) were used. Plates were incubated overnight at 37°C and CFUs counted the next day. For C. jejuni enumeration, serially diluted and homogenized tissues were plated onto Campylobacter-specific agar plates containing Karmali selective supplements (Oxoid) and incubated under microaerobic conditions for 24–48 h at 37°C [59 (link)]. Bacterial counts were normalized to tissue weight.

Polar metabolites were extracted from 100 mg of ¹³C algal lyophilized cells (Synechococcus sp.; Sigma-Aldrich) in a conical tube using a double Folch extraction method (40 (link)): On milliliters of 2:1 chloroform:methanol was added to the sample and subsequently vortexed and sonicated as described above. Next, 400 μl of H₂O was added, along with a metal bead (QIAGEN), and the sample was placed in a tissue lyser (SLS) according to the manufacturer’s recommendations. Afterward, the sample was centrifuged at maximum speed for 10 min at 4°C. The upper, aqueous phase containing, among others, polar metabolites was then transferred to a new conical tube, and the extraction was repeated. Last, the aqueous phase was dried under nitrogen as described above, and the dried sample was reconstituted in 240 μl of 80% methanol, including 1.7 μg of norvaline/ml and stored at −20°C. All solvents used were of LC-MS–grade purity.