K2edta microtainer tubes

Tumour-bearing mice were randomized and assigned into groups (n = 3–10 per group). Vehicle or RMC-7977 was administered via oral gavage daily at 10 mg kg⁻¹ and mice were treated for 21–28 days. Studies were terminated early if tumour burden reached humane endpoint. Body weights were collected twice a week during the study. Means ± s.e.m were plotted in the waterfall plots. For the single-dose pharmacokinetic–pharmacodynamic study, mice were randomized and assigned into groups (n = 3–6 per dose and timepoint). A single dose of RMC-7977 was administered orally at 10 mg kg⁻¹, 25 mg kg⁻¹ or 50 mg kg⁻¹. Tissues (including tumour, colon and skin) were collected at indicated timepoints and either fixed in 10% formalin, embedded in Optimal Cutting Temperature (OCT; Sakura, 4583) solution or snap-frozen in liquid nitrogen for further analysis. Whole blood was transferred into K₂EDTA Microtainer tubes (BD, 365974), incubated for 5 min and snap-frozen in liquid nitrogen.

Mice were infected via gastric intubation with ST (5 × 10⁶ cfu) and blood was collected into K₂ EDTA microtainer tubes (BD Biosciences) and mixed on a rotator for 10 m at room temperature. Plasma was collected after centrifugation at 13,000 rpm for 10 m. Neuraminidase (Neu) activity was detected via Amplex Red (Thermo Fisher Scientific) using 25 µL plasma as per manufacturer recommendations. Control Neu activity was determined using a neuraminidase standard (Arthrobacter ureafaciens; EY Laboratories) serially diluted 2-fold in PBS starting at 500 U/L.

Inocula of S. Typhimurium strains D23580, a multilocus sequence type (ST) 313 strain isolated from blood, SL1344, an ST19 isolate from a calf with salmonellosis [19 (link)] and SARA16, an ST19 human isolate and reference strain [20 (link), 21 (link)], were cultured in LB with shaking for 16–18 hours at 37°C. The identity of each strain was confirmed by antibiotic resistance profiling. Mice received 100 μl of sterile PBS or 100 μl of 1000 colony-forming units (CFU) diluted in sterile PBS by intraperitoneal (i.p.) injection. Mouse weights were monitored daily during infection. Mice were euthanized if they showed signs of lethal morbidity. Systemic bacterial levels were characterized at day 1, 4 or 5 post-infection by determining tissue loads of S. Typhimurium (CFU). Liver and spleen were collected in PBS, weighed and homogenized using an Ultra Turrax T25 Basic mixer from IKA (Staufen, Germany). Blood was collected in K2 EDTA microtainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ), kept on ice for one hour, and then incubated for 10 minutes with 100–200 μl of 1% Triton X-100. Liver, spleen and blood samples were serially diluted and plated on LB agar plates containing appropriate antibiotic selection. CFU/gram tissue or CFU/ml was calculated after overnight growth at 37°C.