Precellys 24 dual homogenizer

For total lung RNA preparation, the left lung was flash frozen in liquid nitrogen immediately after dissection. Frozen lungs were homogenized in 2 mL precooled RLT buffer (Qiagen, Hilden, Germany) + 1% β-mercaptoethanol (Sigma-Aldrich) using the Peqlab Precellys 24 Dual Homogenizer and 7 mL-ceramic bead tubes (#91-PCS-CK28L, Peqlab, Erlangen). 150 µL homogenate were then mixed with 550 µL QIAzol Lysis Reagent (#79306, Qiagen). After addition of 140 µL chloroform (Sigma-Aldrich), the mixture was shaken vigorously for 15 s and centrifuged for 5 min at 12,000 xg and 4 °C. 350 µL of the upper aqueous phase (containing RNA) were then further purified using the miRNeasy 96 Kit (#217061, Qiagen) according to the manufacturer’s instructions. After purification, RNA concentration was determined using a Synergy HT multimode microplate reader and the Take3 module (BioTek Instruments, Winooski, VT, USA). RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technologies).

Zebrafish embryos, strain London wild type (LWT), were maintained in E3 medium at 28°C by following standard protocols (17 (link)). Embryos were bred in the aquarium facilities at the University of Sheffield. Microinjection of embryos was performed as described previously (17 (link)). Individual infected embryos were kept in 100 μl E3 medium, and survival was assessed over 90 h. For in vivo complementation experiments, compounds were dissolved in E3 medium and were buffered to a pH of 6.5 to 7.5. Immediately following injections, embryos were placed in appropriate compound solutions. Further compound solution was added in the embryo washing step. Ninety-six-well microtiter plates were placed in a plastic box, with damp paper, to reduce evaporation during incubation.
pu.1-antisense morpholino-modified oligonucleotides (49 (link)) were injected into zebrafish embryos using the method described previously (17 (link)). Bacteria were recovered from infected embryos at 12-h intervals. Individual embryos were transferred to microcentrifuge tubes and were homogenized using a PreCellys 24-Dual homogenizer (Peqlab). Bacterial numbers were then determined by serial dilution in phosphate-buffered saline (PBS) and plating onto BHI agar.

For biochemical analyses brains were separated into ipsi- and contralateral hemispheres, snap-frozen on dry ice, and stored at −80 °C. Brain samples were homogenized in ice-cold Ca²⁺- and Mg²⁺-free PBS (pH 7.4) in the presence of phosphatase and protease inhibitors (HALT inhibitor cocktail; Thermo Fisher Scientific) by two 30-s cycles at 6500 rpm in a Precellys 24-Dual homogenizer (Peqlab) to reach a final concentration of 10% (w/v). Total protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fischer Scientific).