Microloader tip

Zebrafish experiments were performed as approved by the local animal ethics committee (OPBA, Organismo Preposto al Benessere degli Animali, Università degli Studi di Brescia, Italy). Embryos from the Tg (fli1:egfp) strain of the zebrafish, Danio rerio, were collected, staged, and raised at 28.5 °C, according to standard experimental conditions. Embryos at 48 h post-fertilization (hpf) were anesthetized using 0.04 mg/mL of tricaine (Sigma-Aldrich, St. Louis, MO, USA), and placed onto an agarose gel plate meld for tumor cell microinjection. MM.1S PTX3 cells cultured for 48 h in the presence or absence of DOXA (200 ng/mL) were washed and transferred using a micro-loader tip (Eppendorf, Milan, Italy) into a borosilicate glass needle (outer diameter/inner diameter: 1.2/0.68 mm) connected to a FemtoJet microinjector and InjectMan NI 2 Micromanipulator (Eppendorf). Finally, tumor cells were injected into the perivitelline space of embryos under a stereo-dissecting microscope (Leica, MZ75). Twenty-four hours after tumor injection, the angiogenic response was analyzed by quantifying the cumulative length of sprouts originating from the subintestinal vein vessels after phosphatase staining.

Subjects underwent three test sessions for capillary blood and urine samples collection: the first (Pre-Race) was performed 1–2 days preceding the race in Courmayeur, the second (Middle-Race, 148.7 km) at an almost intermediate point in Donnas, the last (Post-Race) immediately followed the end of the race in Courmayeur. In every session (Pre, Middle and Post Race), for each recruited subject, capillary blood (480 μL): was taken from the fingertip: ROS production rate, antioxidant capacity, lactate, glucose concentrations and hematocrit were determined. Plasma samples were obtained by centrifugation of heparinized capillary blood: plasma was aspirated by using micro-loader tips 0,5–20 μL (Eppendorf, U.S.A.) The samples were collected in triplicate. Urine samples were collected Pre and Post race by voluntary voiding in a sterile container provided to the subjects. All samples were stored in multiple aliquots at -80°C until assayed. Samples were thawed only once before analysis, performed within two weeks from collection.

Worms used for egg laying were kept in synchronized groups of roughly 500 per plate and transferred twice per week to prevent mixing with newly hatching offspring. The day before microinjections, around 1000 worms from 2 plates were combined (to increase the number of eggs laid per plate) and transferred to plates with fresh f/2 medium and no food (to remove the leftover food from the digestive tracks of the animals as food debris can attach to the eggs and impair the microinjections by clogging needles and sticking to holders). On the day of the injections, worms were once again transferred to fresh f/2 without food to remove any debris and eggs laid overnight. Worms were kept in the dark for 3 h and then transferred to light. After 30 min in the light, eggs were collected using plastic pickers made from microloader tips (Eppendorf, Germany), placed on a glass slide in a drop of f/2 and aligned in a line for easier handling.