Omni th homogenizer

Heart protein lysate was obtained by granulation of heart tissue in liquid nitrogen and subsequent resuspension in T-PER Tissue Protein Extraction Reagent (Thermo Scientific, Rockford, IL, USA), containing 1× Halt Protease and phosphatase inhibitor cocktail (Thermo Scientific). The suspension was homogenized using Omni TH homogenizer (Omni International, Kennesaw, GA, USA), and homogenate was cleared by centrifugation (13 500×g/10 min at 4 °C). Protein concentration was measured using the Pierce BCA Protein Assay (Thermo Scientific) according to the specifications supplied by the manufacturer. Myeloperoxidase (MPO) activity was analyzed using the EnzChek Myeloperoxidase (MPO) Activity Assay Kit (Invitrogen), according to the instructions by the supplier. In brief, a total of 0.5 µg protein lysate was added 50 µl Amplex UltraRed substrate and incubated 30 minutes at room temperature. Fluorescence was measured after 30 minutes using Synergy H1 hybrid reader (BioTek, Highland Park, Illinois, USA).

Supernatant. Frozen supernatant samples were allowed to thaw and the protein was precipitated by adding 20% trichloroacetic acid (TCA) and incubated at − 20 °C overnight. The following day the samples were thawed and centrifuged at 4500 xg at 4 °C for 20 mins to pellet the protein. The supernatant was decanted and the protein pellet was washed 2 times with ice-cold acetone. The pellet was allowed to slightly dry and 100 μl of UPX Universal Protein Extraction buffer (expedeon, San Diego, CA) was added and water-bath sonicated into solution. Each sample was incubated at 95 °C for 5 mins to ensure reduction and solubilization of protein. The samples were then vortexed and sonicated for 2 mins, lightly spun to collect condensate and allowed to cool at 4 °C for 45 mins. The samples were then centrifuged at 15,000 xg for 10 mins.
Fungal Pellet. TissueLyser II system (Qiagen, Valencia, CA) trays were frozen at − 20 °C overnight. Two 3 mm stainless steel beads were added to each sample tube and placed in the TissueLyser, the frozen samples were ground for 2 mins at 30 Hz until powderized.
1 mL of UPX extraction buffer was added to each sample and a hand-held OMNI TH homogenizer (OMNI International, Kennesaw, GA) was used to homogenize the sample for 5 mins on ice. Aliquots (1 mL) of each homogenate were removed into fresh tubes and spun at 5000 xg for 10 min.

Co-challenge experiments with CBA/J mice were carried out as described previously (Johnson et al. 1987 (link)) using a modification of the Hagberg et al. (1983 (link)) protocol. Briefly, bacteria were cultured overnight in 5 mL LB, diluted to an OD₆₀₀ of ∼0.2, mixed 1:1, and mice were inoculated transurethrally with 50 μL of 2 × 10⁸ CFU mL⁻¹ (1 × 10⁷ CFU per mouse). Mice were euthanized 7 days postinoculation and urine, bladder, and kidneys were harvested and transferred into sterile tubes containing 3 mL phosphate-buffered saline (0.128 mol/L NaCl, 0.0027 mol/L KCl, pH 7.4). Tissues were homogenized using an Omni TH homogenizer (Omni International Kennesaw, GA, USA) and plated onto LB agar using an Autoplate 4000 spiral plater (Spiral Biotech). Colonies were enumerated with a QCount automated plate counter (Spiral Biotech). A competitive index (CI) was calculated for each organ with a bacterial load greater than the limit of detection by determining the ratio of mutant to wild type as follows:
A CI of 1 indicates that the mutant colonized the organ to a similar level as P. mirabilis HI4320, a CI <1 indicates that the mutant was outcompeted by wild type, and a CI >1 indicates that wild type was outcompeted by the mutant.

Human brain
tissue (1 g) was homogenized in 5 mL of TTL lysis buffer (50 mM Tris-HCl
pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM Na₃VO₄, 20 mM NaF, 1 × protease inhibitors complete
EDTA-free, all from Sigma-Aldrich, Saint-Louis, U.S.A.) using an OMNI
TH homogenizer (Omni International, Kennesaw, U.S.A.) at medium speed
for 2 min at 4 °C. The homogenate was centrifuged at 30 000g for 20 min at 4 °C. The supernatant was transferred
into a fresh tube. The pellet was homogenized in the same volume of
TTL lysis buffer and under the same conditions. After centrifugation,
both supernatants were pooled.

PR8 virus TCID₅₀ infectivity value and lung tissue viral burden were determined as endpoint serial dilutions of virus or tissue homogenates at which 50% of the cells were infected using Madin-Darby canine kidney (MDCK) cells. Lungs obtained from animals 2, 3, or 4 days after INFV A exposure were homogenized using an Omni TH Homogenizer (Omni International, Kennasaw, GA, USA) and Omni tissue disposable generators in Lebovitz L-15 medium (Gibco Life Technologies, Grand Island, NY, USA) containing antibiotic-antimycotic mixture (Invitrogen, Grand Island, NY, USA). After clarification by centrifugation, supernatant was added (20 µL/well) to 4 replicate wells of a 96-well flat-bottomed tissue culture plate (Corning, Tewksbury, MA, USA) containing MDCK cells in logarithmic (log) phase growth, and serially diluted 10-fold for a total of 8 dilutions. The plates were incubated at 37 °C in 5% CO₂ for 4 days and cells inspected microscopically. Cytopathic effect was noted and confirmed visually by adherence of turkey RBC. The results of viral load assessment were expressed as TCID₅₀ per gram of tissue [24 (link)].
To quantify lung viral load, four mice per group were euthanized on day 3 following PR8 H1N1 virus challenge and on days 2 and 4 following pH1N1 or H5N1 virus challenge.