Magna lyser homogenizer

In order to determine the impact of three different methods on viability of planktonic bacteria, suspensions were subjected to homogenization or bead beating for various times.
Staphylococcus aureus and Escherichia coli previously isolated in the Biomaterials-Related Infection laboratory, University of Nottingham, UK, were used for the preparation of bacterial suspensions in tryptic soy broth (TSB, Oxoid, Basingstoke, UK) at a concentration of 10⁵ cfu/mL. Concentration was controlled by light absorbance calibration and plating of serial dilutions.
One hundred microliters of each suspension was added to 1 mL of phosphate-buffered saline (PBS) and treated by either homogenization for two or four cycles (each cycle lasting for 45 s at a speed of 4500 rpm with 45 s between cycles) or bead beating for two or four cycles in the same way as homogenization using 0.1-mm glass beads. The MagNA Lyser homogenizer (Roche, Basel, Switzerland) was used.
One hundred microliters of bacterial suspensions were also added to 0.1% w/v dithiothreitol (DTT, Thermo Fisher Scientific, Vilnius, Lithuania) in sterile water for 5 min before plating to be compared to homogenized samples for four cycles as described above.
One hundred microliters of each of processed and control (unprocessed) bacterial suspension was spread on blood agar plates (Oxoid), and colonies were counted after overnight incubation.

Immunoprecipitation and western blot analyses were performed as described earlier^{17 (link)}. Briefly, livers or lungs from ob/ob mice were homogenized in 1 mL of lysis buffer with a MagNa lyser homogenizer (Roche, Indianapolis, IN). 5 mg of total liver lysates were immunoprecipitated with 10 μl of sepharose-conjugated anti-Akt or phospho Stat3 antibodies and immunoblotted with an anti-phospho Akt or anti-Stat3 rabbit polyclonal antibodies, respectively. Total Akt and Stat3 in the liver lysates (50 μg) were detected using an anti-Akt and Stat3 rabbit polyclonal antibodies, respectively. ERK1/2 phosphorylation studies in HepG2 cells were performed by immunoblotting for phospho ERK1/2 and anti-ERK1/2 with rabbit polyclonal antibodies, as described^{2 (link)}. All antibodies were purchased from Cell Signaling (Danvers, MA). mBP3 expression in transfected ob/ob mouse lungs was detected by immunoblot analysis using an anti mouse FGFBP3 rabbit polyclonal antibody (Abgent).

Total RNA samples were prepared using the RNeasy Plus Micro Kit (#74034; Qiagen, Hilden, Germany). Briefly, gastric mucosa tissues were homogenized in Buffer RLT Plus using the MagNA Lyser homogenizer (Roche, Mannheim, Germany). The lysates were centrifuged at full speed for 3 min, the supernatant was carefully transferred to a gDNA Eliminator spin column, and RNA was isolated according to the manufacturer’s protocol. mRNA was reverse-transcribed using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, San Diego, CA, USA), and qPCR was performed using a specific primer and SensiFAST SYBR Lo-ROX Kit (Bioline, Taunton, MA, USA) on a LightCycler 480 System (Roche, Mannheim, Germany). Primer sequences used in this study are listed in Table S1. The relative mRNA expression of each gene was normalized to that of the housekeeping gene (L32) and calculated using the 2^−ΔΔCT method.

Vero cells were plated at 2x10⁵ cells per well in 12-well plates and allowed to grow to 90–95% confluency overnight. Superior and middle lung lobes were homogenized in 0.5 mL of media (DMEM + 5% FBS + 1 mM L-glutamine) at 6000 rpm for 40 sec using a Roche MagNA Lyser homogenizer and centrifuged for 1 min at full speed to pellet debris. 50μL of the supernatant was added to 450 μL DMEM + 5% FBS + 1 mM L-glutamine media, and ten-fold serial dilutions were made to create a dilution series of 10⁻¹ to 10⁻⁶. 200 μL of each homogenate dilution were added to the plated Vero cells and incubated at 37°C. After 1 h, 2 mL of overlay (50:50 mixture of 2.5% carboxymethylcellulose and 2X alpha MEM + 6% FBS + 2% penicillin/streptomycin + 2% L-glutamine + 2% HEPES) was added to each well, and plates were incubated at 37°C, 5% CO₂ for 4 days. 2 mL of 4% paraformaldehyde was added to each well and allowed to fix cells overnight. Following removal of the fixative, wells were stained with 0.25% crystal violet, and visible plaques were counted and averaged between two technical replicate wells. Viral titers were calculated as pfu per lung tissue. The limit of detection (LOD) for the assay was determined to be 12.5 pfu / lung tissue, and samples that yielded no plaques were assigned a value of 6.25, half of the LOD.

To assess bacteremia (blood circulating bacteria) in infected animals, 10 µl of blood was sampled before infection, 5 min after infection, and at the time of sacrifice. Serial dilutions of blood were plated on GCB agar plates and incubated overnight at 37 °C and in a moist atmosphere containing 5% CO₂. Bacterial counts were expressed in colony-forming units (CFU) per ml of blood.
To assess the extent of vascular colonization by meningococci (adherent bacteria) following mouse sacrifice at indicated times post infection, tissue biopsies were collected using a sterile dermatological biopsy puncher (~4 mm²), weighted and placed in 500 µl 1× PBS. Skin biopsies were dissociated and homogenized using MagNA lyser homogenizer (Roche, France) and serial dilutions of skin homogenates were plated on GCB plates and incubated overnight at 37 °C and in a moist atmosphere containing 5% CO₂. Bacterial counts were expressed in colony-forming units (CFU) per mg of the skin.

Harvested wound tissues were placed in tubes pre-filled with homogenizer beads (Roche, Indianapolis, IN). After adding 1 mL of PBS, the tubes were homogenized for 90 s at 5000 rpm in a MagnaLYSER homogenizer (Roche, Indianapolis, IN). The homogenate was removed for determination viable counts (500 μl) and for isolating bacterial genomic DNA for determinations of Pseudomonas and total bacterial counts by PCR (200 μl). The viable counts were determined as previously described [3 (link)], and control wounds were similarly assayed to ensure their sterility.
For the determination of total cell counts by PCR, bacterial DNA was isolated from 200 μl of the homogenate (cleared by centrifugation, 3 min at 4°C at 14,000 rpm in an Eppendorf 5417R), using the DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA), according to the manufacturer's instruction. Total P. aeruginosa cell counts were quantified by PCR analysis of the outer membrane lipoprotein gene oprL [42 (link)]. Total bacterial load was quantified by PCR analysis of 16S rDNA [43 (link)]. The primers and probes were synthesized by Applied Biosystems (Carlsbad, CA) and were listed in S3 Table. For these determinations, the standard curve was generated using the genomic DNA that was isolated from mid-log growth phase P. aeruginosa cultures (quantified using the Quant-iT ds DNA BR Assay Kit, Invitrogen).

RNA extractions, reverse transcription and qPCR were performed according to the manufacturer’s specifications and described by Fröhlich et al. (2016) (link). Hypothalamic tissue samples were homogenized with the MagnaLyser homogenizer (Roche Diagnostics). RNA was extracted using the RNeasy lipid tissue mini kit (Qiagen, Hilden, Germany). Subsequently, RNA concentrations were determined using NanoDrop (Thermo Fisher Scientific, Vienna, Austria), and 2 μg of RNA of each sample was reverse-transcribed in the Mastercycler Gradient (Eppendorf, Hamburg, Germany), using the high capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Afterward, mRNA levels were quantified in triplicates via qPCR using a LightCycler 480^® system with TaqMan gene expression assays for IFN-γ (Mm01168134_m1), IL-1β (Mm00434228_m1), IL-6 (Mm00446190_m1), NPY (Mm03048253_m1), and TNF-α (Mm00443258_m1) and with the TaqMan gene expression master mix (Thermo Fisher Scientific). Negative controls without reverse transcriptase added were included for each treatment group. As reference genes (endogenous housekeeping), PPIL3 (Mm00510343_m1), and ACTB (Mm00607939_s1) were used. In order to quantitate target gene levels relative to controls the 2^-ΔΔCt method was used, where the mean value of the vehicle treated group was used as calibrator and group differences were expressed as fold changes.