Mononylon 5

Mice were joined in parabiosis as previously described.³⁹ Mice were anesthetized with 2% isofluorane (2L/min O2). Briefly, after shaving the corresponding lateral aspects of each mouse, matching skin incisions were made from behind the ear to the tail of each mouse, and the subcutaneous fascia was bluntly dissected to create ~0.5 cm of free skin. The scapulas were sutured using mono-nylon 5.0 (Ethicon, Albuquerque, NM), and the dorsal and ventral skins were approximated by continuous suture. All mice undergoing parabiosis surgeries were injected twice daily with buprenorphine (0.1mg/kg i.p.) for three days beginning on the day of surgery. Mice were joined for intervals of 2 weeks before proceeding with coronary ligation.

The procedure was conducted as previously described^{10 (link)}. In brief, age-, sex, and weight-matched animals were used and housed together for a least 14 days prior to surgery. The corresponding lateral aspects of each mouse were shaved, incisions were made from the forelimb joint to the hindlimb joint and the subcutaneous fascia was bluntly dissected to create 0.5 cm of free skin. Fore- and hindlimb joints were joined and the dorsal and ventricle skins were approximated by continuous suture using mononylon 5.0 (Ethicon).

The procedure, adapted from was conducted as previously described^{47 (link)}. Briefly, after shaving the corresponding lateral aspects of a GFP-UBI and a wild type mouse, matching skin incisions were made from behind the ear to the tail of each mouse, and the subcutaneous fascia was bluntly dissected to create about 0.5 cm of free skin. The scapulas were sutured using a mono-nylon 5.0 (Ethicon, Albuquerque, NM), and the dorsal and ventral skins were approximated by continuous suture. Mice were joined for 2 weeks. After, mice in parabiosis were surgically separated by a reversal of the procedure. Percent chimerism in the blood was defined for gated Ly-6C^hi monocytes as %GFP+ (%GFP⁺ & %GFP⁻)⁻¹ in wild type mice. Mice in the fasting group were fasted directly after separation for 28 hrs and mice in the refed group refed after 24h for 4h.

Cannula and osmotic minipump (Alzet) implantation were performed as previously described^{11 (link)}. Briefly, mice were anesthetized, the head was shaved and secured in a stereotactic frame (Stoelting). An incision was made above the skull extending behind the shoulder blades. A small hole was drilled in the skull at AP −1; ML −0.27 from bregma and depth 2mm from dura to target the lateral ventricle. The cannula was inserted and glued to the skull. The cannula was connected to an osmotic minipump filled with recombinant IL-3 (Biolegend) conjugated to an anti-IL-3 antibody (Biolegend) as previously described³ . Minipumps delivered rIL-3 into the ventricle at a rate of 1μ/day. Minipumps were implanted subcutaneously caudal the shoulder blades. At the end of the procedure the incision was sutured using mononylon 5.0 (Ethicon).