Macs octo dissociator with heaters

To induce muscle injury, mice were anesthetized with isoflurane, and cardiotoxin (CTX) from Naja pallida (10 μM, Latoxan Laboratory) was injected directly into the tibialis anterior (TA) muscle (40 μl) as well as into the gastrocnemius muscle (80 μl). CTX was injected once, and mice were euthanized 5 days after CTX injection. 3 h before sacrifice, mice were injected intraperitoneally with EdU (Invitrogen) (10 mM in PBS, 10 μl/g body weight). Hindlimb muscles were dissected, minced, and dissociated in a collagenase/dispase solution using the gentle MACS Octo Dissociator with Heaters (Miltenyi Biotec). Cells were filtered, centrifuged and resuspended in FACS buffer (10%FBS, 3 mM EDTA in PBS). For proliferation assays, cells were stained with α7-INTEGRIN and lineage markers (CD31, CD11b, SCA-1 and CD45) (listed in Supplementary Table 2), then fixed using BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences). EdU was then visualized using Click-iT EdU Imaging Kit (Invitrogen) according to the manufacturer’s instructions. Cells were then incubated with propidium iodide (20 μg/ml) and RNAse A (0.5 μg/ml) for 16 h before analysis. Analysis was performed on a BD LSRFortessa instrument using the DIVA software.

Blood cells were obtained from the mice using a microtube containing EDTA (Erma Inc., Tokyo, Japan). The spleens were minced onto glass slides. BM cells were collected by flushing the femurs and tibias with a 25-gauge needle. The livers and lungs were minced and processed using a gentle MACS Octo Dissociator with Heaters (Miltenyi Biotec, Bergisch Gladbach, Germany). Supernatants were filtered using a MACS SmartStrainer (pore size: 100 µM; Miltenyi Biotec) and centrifuged at 300×g for 10 min. The pellet was resuspended in Debris Removal Solution (Miltenyi Biotec) and centrifuged at 3000×g for 10 min. The cell pellet was resuspended in red blood cell (RBC) lysis buffer (BioLegend).

Male Il13^+/+, Il13^+/- and Il13^-/- BALB/c mice received daily injections of rIL-33 (0.5 µg, i.p.) for 5 days. The mice were euthanized and perfused with PBS, and then liver and lung ILC2s were harvested. Briefly, the mouse livers and lungs were digested using a gentle MACS Octo Dissociator with Heaters (Miltenyi Biotec) using a liver dissociation kit (Miltenyi, 130-105-807, program: 37C_m_LIDK_1) and a lung dissociation kit (Miltenyi, 130-095-927, m_lung_01, incubation at 37 °C for 30 min, and then m_lung_02 program), respectively. Debris was removed using the protocol published by Itoh, with some modifications⁴⁰ . After filtration through a 70-µm cell strainer, the cells were collected by centrifugation at 500 × g for 5 min at 4 °C and resuspended in 40% Percoll Plus (GE Healthcare). Then, 75% Percoll was gently added below the 40% Percoll layer. After centrifugation at 700 × g for 20 min at RT, the cells at the interface were collected in PBS containing 1% FBS and 2 mM EDTA, and lineage-positive cells were depleted using MACS (Miltenyi) with an anti-mouse lineage panel (BioLegend, 133307) and streptavidin nanobeads (BioLegend, 480016). Then, single-cell suspensions were stained using blocking and staining antibodies in the dark at 4 °C. Liver Lin^-Th1.2⁺IL-7Rα⁺ST2⁺ ILC2s were sorted using a FACSAria cell sorter (BD Biosciences) and cultured or directly used for experiments.