Ceramic sphere

The mice were humanely euthanized on day 3 or day 7 p.i., and spleen, liver, ileal, colon, and, cecal tissue samples were collected and processed for total Campylobacter load. The tissue samples were placed into 0.5 mL of 0.9% saline in sterile lysis matrix tubes containing ceramic spheres (1.4 mm; MP Biomedicals, USA). Additional spleen, liver, and ileal samples were collected in RNAlater solution (Invitrogen, Australia) and stored in a −80°C freezer for RNA extraction.
The tissue samples were homogenized using a FasTPrep-24 5G sample preparation system (MP Biomedicals, USA) at 6.0 m/s speed for 40 s. Serial 10-fold dilutions of the homogenates were prepared, and 100 μL of each dilution was spread plated onto mCCDA agar and incubated at 42°C for 48 h in 10% CO₂. Subsequently, 100 mg of fecal pellets was suspended in nutrient broth no. 2 (Thermo Scientific, Australia) and incubated at 42°C for 48 h in 10% CO₂, followed by spread plating onto mCCDA. All culture-negative tissue samples and fecal pellets were resuscitated by enriching in nutrient broth no. 2, subsequent incubation at 42°C in 10% CO₂ for 24 h, and plating onto mCCDA.

The mice were humanely euthanized on day 3 or day 7 p.i., and spleen, liver, ileal, colon, and, cecal tissue samples were collected and processed for total Campylobacter load. The tissue samples were placed into 0.5 mL of 0.9% saline in sterile lysis matrix tubes containing ceramic spheres (1.4 mm; MP Biomedicals, USA). Additional spleen, liver, and ileal samples were collected in RNAlater solution (Invitrogen, Australia) and stored in a −80°C freezer for RNA extraction.
The tissue samples were homogenized using a FasTPrep-24 5G sample preparation system (MP Biomedicals, USA) at 6.0 m/s speed for 40 s. Serial 10-fold dilutions of the homogenates were prepared, and 100 μL of each dilution was spread plated onto mCCDA agar and incubated at 42°C for 48 h in 10% CO₂. Subsequently, 100 mg of fecal pellets was suspended in nutrient broth no. 2 (Thermo Scientific, Australia) and incubated at 42°C for 48 h in 10% CO₂, followed by spread plating onto mCCDA. All culture-negative tissue samples and fecal pellets were resuscitated by enriching in nutrient broth no. 2, subsequent incubation at 42°C in 10% CO₂ for 24 h, and plating onto mCCDA.

At 10 days after the intravenous injection of Miltuximab^®-IR800 or IgG-IR800, the mice were sacrificed by isoflurane overdose with subsequent cervical dislocation. The tumors and major organs were then collected, imaged using the Odyssey CLx fluorescent imager, and preserved by freezing for subsequent analysis. In order to avoid the effect of possible attenuation of light on ex vivo imaging of whole tumors and organs of variable thickness, the samples were later homogenized and analyzed as was described by Oliveira et al. [57 (link)]. Briefly, a sample of 50–150 µg of each tumor and organ was collected and homogenized. For the homogenization, the samples were suspended in 800 µL RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-11, 0.1% SDS, and 0.5% sodium deoxycholate) in a tube with a 1/4 in. Ceramic Sphere and 1.4 mm Ceramic Beads (MP Biomedicals, Santa Ana, CA, USA). The samples were then disrupted by a FastPrep-24 homogenizer (MP Biomedicals Santa Ana, CA, USA) at 40 sec, 5 min off, and three times at room temperature. The samples were then serially diluted with RIPA buffer in a clear bottom 96-well plate, and imaged with the Odyssey CLx.

DNA was extracted from ticks by a standard protocol using the phenol/ethanol method (15 (link)). The spleens of the wild rodent were shred to 100 mg pieces and homogenized in a tube containing a ceramic ball (Ceramic Sphere, MP Biomedicals; catalog no. 6540412), beads (Garnet Matrix A Bulk, MP Biomedicals; catalog no. 6540–427), and TNE buffer (50 mM Tris–HCl [pH 7.4], 100 mM NaCl, and 0.1 mM EDTA) by shaking at 4,000 rpm for 30 s (Micro Smash MS-100, TOMY). Proteinase K (0.1 mg/mL) and SDS (0.1%) were added and incubated at 55°C overnight. DNA was extracted using the standard phenol/ethanol method, dissolved in 200 µL TE (10 mM Tris–HCl, pH 7.4, 1 mM EDTA), and stored at −30°C.

Skin swabs were collected from shaved back skin of four Spink5 cKO mice and two control mice on the same day. Staphylococcus aureus (S.aureus) was detected by quantitative PCR according to procedures and protocols at Charles River Laboratories. The estimated DNA copy number for each S.aureus assay was reported. For quantification of bacterial load in skin, blood and internal organs (spleen, liver, lungs) of WT control and Spink5 cKO mice, tissues were mixed with 0.5 mL of sterile PBS in 2 mL lysing matrix D tubes each containing one ¼” ceramic sphere (MP Biomedicals). Tissues were homogenized using FastPrep-24 tissue homogenizer (MP Biomedicals) for 3 shake cycles of 20 seconds each. The homogenates were spread on pre-warmed Brain and Heart Infusion agar and mannitol salt agar plates (100 µL of homogenate per plate). Skin homogenates were diluted 1/50 in sterile PBS before plating. Plates were incubated at 37 °C in aerobic conditions for 48 h. Colonies were counted and CFUs/g values were calculated considering the weight of the harvested tissue samples, the volume of homogenate spread per plate and the dilution factor (in case of skin samples). Mannitol salt agar plates contained 5% v/v egg yolk emulsion.