Microcentrifuge tube

The clinical MMEs were performed by trained laboratory technicians at the PWHC following a positive dipstick test. The SIEMENS Clinitek Status+ was used to read the Multistix 10 SG reagent strips (ref. 2161), and a positive test for blood, leukocyte esterase, or nitrate resulted in an MME being performed. To prepare the specimens for microscopy, a 1.5 mL microcentrifuge tube (VWR, Radnor, PA, USAmanufacture, city, state abbreviation if available, country) was filled with urine and centrifuged at 9814 RPM for 45 sec. The supernatant was decanted, and the remaining droplet was mixed to resuspend any cells on the bottom of the tube. As an optional step, Sternheimer Malbin (Astral Diagnostics 6561-01, Paulsboro, NJ, USAcity, state abbreviation if available, country), a contrast agent, was mixed with the cell sample to provide contrast for the MME. A plastic pipette was used to transfer the cell sample into a plastic cassette to form a monolayer of cells. A transmission microscope with a 40× Olympus 0.65 Plan Air objective lens (Olympus, Shinjuku, Tokyo, Japancity, state abbreviation if available, country) in phase contrast was used to examine the cell sample. Cells were manually counted in 9 FOVs (total area of 0.134 mm²) and averaged. Results were reported as reference ranges rather than absolute values, for instance, “Few (3–10)”.

3 × 10⁷ MV4-11 or THP-1 cells were seeded into suspension culture flasks for each treatment group (vehicle control, 1 µM artesunate and 10 µM artesunate). Cells were treated for 24 h at 37 °C with 5% CO₂, prior to isolation for both cytoplasmic and mitochondrial fractions using the Mitochondrial Isolation Kit (Sigma Aldrich, cat# MITOISO2). Following treatment cells were washed in PBS, the cell pellet was resuspended in 1.8 mL extraction buffer A, and cells were incubated on ice for 15 min. Next cells were homogenized using 30 strokes with a 2 mL Dounce homogenizer using the tight pestle (Sigma-Aldrich). Cell debris was removed by centrifugation at 600 × g for 10 min at 4ºC. The supernatant was transferred to a new microcentrifuge tube (VWR) and centrifuged at 11,000 × g for 10 min at 4 °C to separate the cytoplasm from the mitochondrial pellet. The supernatant was transferred to a new microcentrifuge tube labeled as the cytoplasmic fraction and was stored at − 20 °C for use in cytochrome c ELISA assays (details below). The pellet which contains intact mitochondria was resuspended in 200µL 1 × storage buffer and kept on ice for immediate use in cytochrome c oxidase activity assays (details below).

Immobilisation of biotinylated primary CRP antibodies (1° anti-CRP) onto streptavidin functionalised magnetic particles (Dynabeads M-270 Streptavidin) was achieved via the streptavidin-biotin interaction, as previously reported [69 (link),70 (link)]. Briefly, 10 µL of stock particle suspension (6.5 × 10⁸ particles·mL⁻¹) was added to a 1.5 mL microcentrifuge tube (VWR, Leicester, UK), followed by 200 µL of 1° anti-CRP solution at a concentration of 10 µg·mL⁻¹, and incubated for 15 min with slow tilt rotation in order for the biotinylated antibodies to bind to the streptavidin-coated particles. The particles were then washed three times using the following procedure.
The particles were pulled to the side of the tube via an external magnet and the supernatant removed using a pipette. PBS solution (1000 μL) was added to the tube, which was vortexed for 20 s to resuspend the particles. This washing process was repeated twice more, and the particles finally resuspended in PBS buffer solution.

To quantify fluralaner and fenbendazole in the chicken serum, 2.5 ml of whole chicken blood was collected in 3.0-ml BD Vacutainer® blood collection tubes (BD Manufacturing, Glenboro, MB, Canada) and then centrifuged at 10,000 RPM for 20 min, following which 1.0 ml of serum was transferred into microcentrifuge tubes (VWR International, Radnow, PA, USA). Serum samples were stored for approximately 3 months at − 20 °C before testing. Targeted liquid chromatography tandem mass spectrometry (LC-QQQ) analysis was performed on a TSQ Quantiva mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a binary pump UHPLC (Ultimate3000; Thermo Fisher Scientific). Scan parameters for target ions in fluralaner and fenbendazole are given in Table 1. Chromatographic separation was achieved on a Hypersil Gold 5 µm, 50 mm × 3-mm C18 column (Thermo Fisher Scientific) maintained at 30 °C, using a solvent gradient method. Sample acquisition and data analysis were performed using Trace Finder 3.3 application (Thermo Fisher Scientific). Analysis was performed at the Integrated Metabolomics Analysis Core at Texas A&M University.Table 1

Scan parameters for target ions

Insecticide	Polarity	Precursor (m/z)	Product (m/z)
Fenbendazole	Positive	300	131.1
Fenbendazole	Positive	300	159
Fenbendazole	Positive	300	268.1
Fluralaner	Negative	554.1	424.1
Fluralaner	Negative	554.1	494.1
Fluralaner	Negative	554.1	534.1

Isolation of DNA from cow vaginal swabs and calf URT and fecal swabs was performed by adding 1.5 ml of DNA/RNA-free ultra-pure water into the 2-ml microcentrifuge tubes (VWR International, Radnor, PA) that contained URT, feces or vaginal swab samples. The tubes were then vortexed for 10 minutes by using a vortex adaptor (MO BIO Laboratory Inc., Carlsbad, CA) that holds 2-ml microcentrifuge tubes horizontally. The swabs were removed from the microcentrifuge tubes and the remaining liquid was centrifuged for 5 minutes at 13,000 rpm (room temperature). The supernatant was discarded and the DNA was extracted from the pellet using a PowerSoil DNA Isolation Kit (MO BIO Laboratory Inc., Carlsbad, CA) according to the manufacturer’s recommendations.
Isolation of DNA from cow fecal samples was performed after homogenization of the feces content, and a total of 250 mg of feces solution was added directly to the PowerSoil beads tube, and DNA extraction was performed following the manufacturer’s recommendations (MO BIO Laboratory Inc., Carlsbad, CA).
DNA concentration and purity were evaluated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE) at wavelengths of 260 and 280 nm.

Release rates of three different kinds of attractants, including acetoin (AT), ethyl octanoate (EO), and a blend of AT and EO (ratio = 1:1), were measured under controlled conditions in a laboratory fume hood. Each attractant (1 mL) was loaded onto cotton balls held in open micro-centrifuge tubes (1.5 mL, VWR International, Radnor, PA). Fifteen of these micro-centrifuge tubes (N = 5) were suspended on hooks in a fume hood (temperature 20–25 °C, face velocity 129 feet/min). Each tube was weighed using an Ohaus GA110 analytical electronic balance (Pine Brook, NJ) every 24 or 72 h (weekend), and the amount of attractant residue was calculated and recorded over a period of 2 weeks.