Diaphragm Transplantation in mdx Mice

One day prior to transplantation, recipient mice were anesthetized with ketamine/xylazine at 80 mg/kg by intraperitoneal (IP) injection and their diaphragms were irradiated with 10 Gy [67 (link)]. For diaphragm injections, the same ketamine/xylazine combination was used to anesthetize the mice. As previously described [44 (link),45 (link)], a laparotomy was performed to expose the diaphragm. We injected 30 µL of 10 µM CTX (Latoxan, Porte les Valences, France) followed by 5000 FACS-purified SCs resuspended in 30 µL of PBS. Diaphragms injected with PBS alone served as a negative control. Five to eight IM injections were performed in the top right part of the diaphragm muscle using a 33 g Hamilton syringe. For immunosuppression, a dose of 5 mg/kg of the immunosuppressive agent tacrolimus (MedChemExpress, Monmouth Junction, NJ, USA) was injected daily via IP to the immunocompetent mdx^4Cv recipients. Treatment started one day before irradiation and ended by the day of euthanasia [26 (link)]. Diaphragm muscles were collected for engraftment assessment at 4 weeks post-transplantation.