21 gauge needle

The buprenorphine-free control suspension consisted of cholesterol and glycerol tristearate (96:4) suspended in medium-chain triglyceride oil (8 mg/100 uL). The drug suspension consisted of buprenorphine, cholesterol, glycerol tristearate, suspended in medium-chain triglyceride (MCT) oil, and Miglyol 812, (Sasol, Hamburg Germany), 8 mg/100 uL, trade name Animalgesics for Mice. Control and drug suspensions were supplied by Animalgesics Labs (Millersville MD). To limit stress associated with constraining conscious animals for SC injections, rats were anesthetized with an intraperitoneal (IP) solution of ketamine, 80 mg/kg, and xylazine, 8 mg/kg, in a saline solution containing 14.25% ethyl alcohol. Each rat was injected with the designated dose of test article or buprenorphine-free control suspension before they recovered from anesthesia. The dose was administered SC on the mid-dorsal area about 1 cm rostral to the surgical incision using a 21 gauge needle (BD, Franklin NJ) attached to a 1 mL BD Tuberculin syringe. Following dose administration, animals were transferred to a clean cage on a heating pad until recovered. Once the animal regained consciousness and demonstrated normal movement, and with the absence of signs of distress, it was returned to its home cage. Posttreatment distress was not observed.

Crew members warmed and massaged their fingertips to maximize blood flow. Fingertips were sterilized (BZK antiseptic towelette, Dynarex, Reorder No. 1303) and punctured using a contact-activated lancet (BD Biosciences, #366 593) or a 21-gauge needle (BD Biosciences, #305 167). Whatman 903 Protein Saver DBS cards (Cytiva, #10 534 612) were used to capture, transfer, and then store capillary blood with a desiccant pack (Cytiva, #10 548 239) at ambient temperature.

For the Experiment 2 run, each rat was weighed every day, and weight changes were calculated for every group of animals. Immediately after behavioral tests (Experiment 2, day 17), rats were deeply anesthetized with isoflurane, and arterial blood samples were taken by cardiac puncture (with a 21-gauge needle; 0.8 × 40 mm; Becton Dickinson S.A., Madrid, Spain). Additionally, livers were removed, to perform histopathological examination. After that, the animals were all sacrificed.

With the approval from the Ethics Committee of Health Service Center at Seoul National University, we collected blood from healthy volunteers. As gender, age and diseases are all risk factors of thrombosis, we only used healthy male donors ranging from 20 to 30 years old to simplify our study design. Human blood was collected using a vacutainer with acid citrate dextrose (ACD) and a 21 gauge needle (Becton Dickinson, U.S.A.) on the day of each experiments. Platelet rich plasma and buffy coat were removed after centrifugation at 200 g for 15 min. Packed RBCs were washed twice with phosphate buffered saline (PBS: 1.06 mM KH₂PO₄, 154 mM NaCl and 2.96 mM Na₂HPO₄ at pH 7.4) and once with Ringer’s solution (125 mM NaCl, 5 mM KCl, 1 mM MgSO₄, 32 mM HEPES, 5 mM glucose, pH 7.4). Washed RBCs were resuspended in Ringer’s solution to a final cell concentration of 5 × 10⁷ cells/mL with 1 mM CaCl₂ before use.

The volume of SVF cell suspension per knee joint was adjusted to 7 ml using Lactated Ringer and then injected intra-articularly (and into the Hoffa’s fat or IFP) using ultrasound guided injection with a 21 gauge needle (Becton Dickinson).