Conical centrifuge tubes

The collected cells were evenly distributed in five 50 mL Falcon Conical Centrifuge Tubes and the cell membranes were disrupted by sonication at 20 kHz on ice in 3 cycles of 30 seconds separated by 30 seconds pause using a sonicator cell disruptor model W185F (Heat Systems-Ultrasonic Inc.). The lysate was ultracentrifuged using the Beckman Optima LE-80 K for 3 hours at 500,000 g at 4 °C. The supernatant was carefully collected and used for NMN purification by High Performance Liquid Chromatography (HPLC).

Initially, titanium discs were incubated within Falcon^® Conical Centrifuge Tubes (Glendale, AZ, USA) containing FBS-free cell culture medium for 24 h, as recommended by ISO 10993:2016, and the samples were properly centrifuged to avoid any debris and later used to evaluate the behavior of cells (Figure 1). It was here expected that the surface named DAE should release titanium molecules. Furthermore, ECs were exposed to that titanium-enriched medium for 3 days, when the EC-conditioned medium containing angiocrine molecules samples was duly collected to allow the analysis of the titanium content. It is important to consider here that ECs were subjected to shear stress as a testing group, as well as considering non-stressed cells as a comparable and reference group, avoiding the mechanical factors described to mimic hemodynamic forces of physiological blood flow (Figure 1). Both media conditioned by ECs were considered in this study to contain angiocrine molecules, soluble molecules released by ECs from the metabolism in response to soluble titanium. Finally, the angiocrine molecules containing the medium were later used to expose osteoblasts for 7 days, and then the cell samples were harvested to allow for the molecular analysis.

Samples were collected in December 2018 and none of the female cats showed signs of being in estrus (e.g., increased frequency of calling, lordosis, and other sexual behaviors). Unscented nonabsorbent litter (Petconfirm, Nancy Ridge Technology Center, San Diego, CA, USA) was used for urine collection. Fecal samples were collected either from the litter box or with the free-catching method. The researcher checked the litterbox every 30 min during the day (from 8:00 a.m. to 6:30 p.m.) for urine and feces. Contaminated samples were discarded. Urine was collected with centrifuge tubes (Conical Centrifuge Tubes, Falcon^®, Newport, TN, USA) and fecal samples with whirl-pack bags (Sigma-Aldrich, St Louis, MO, USA). Samples were placed on ice immediately after collection, transferred to the lab, and stored at −80 °C until extraction.

The cross-linking reagent sulfo-SDA was purchased from Thermo Scientific Pierce (Rockford, IL). Human blood serum was acquired from a healthy male donor after informed consent, in accordance with standard institutional ethical procedures at the University of Edinburgh, School of Biological Sciences. Immediately following collection (50 ml total volume split over 2× Falcon 50 ml Conical Centrifuge Tubes), blood serum was isolated from the whole blood sample without anti-coagulants, by centrifugation. Whole blood was allowed to clot by leaving it undisturbed at room temperature for 30 min. The clot was removed by centrifuging at 1900 × g for 10 min at 4 °C. The resulting supernatant was immediately apportioned into 1.5 ml Eppendorf Tubes as 0.5 ml aliquots, which were flash frozen using liquid nitrogen and stored in a −80 °C freezer. Protein concentration was estimated at 80 mg/ml using a Bradford protein assay.

Samples (50 g) from the each of the specified intestinal compartments – SI excluded since the compartment did not contain enough material – were collected into Falcon collection tubes (Falcon Conical Centrifuge Tubes, Tewsbury, MA), sealed and frozen and stored at − 20 °C until analysis.
Particle size was determined by wet sieving according to the method described by Vondran et al. [9 (link)]. Briefly, samples were thawed and soaked in beakers containing 1 L water overnight prior to sieving. Samples were passed through sieves of the following mesh sizes for 5 minutes: 8, 4, 2 and 1 mm. The material remaining on each sieve was dried at 60 °C for 12 hours, then cooled before weighing. The dry amount on each sieve was expressed as a percentage of the dry weight of the total sample. The latter was calculated from the weight of the sampled faeces measured before and after drying. The fraction that washed through the finest sieve (< 1 mm) was calculated from the total sample weight minus the sum of the four sieve fractions.