Ly6g v450

Muscle tissue was isolated from the mice and the fascia was sacrificed. Mononuclear cells were isolated from hind limb muscles that were finely minced and enzymatically digested with 0.2% collagenase II as described above in the satellite cell isolation section. The mononuclear cells were stained with the following antibodies: Ly6G-V450 (BD Bioscience, 1:75, clone1A8), F4/80-FITC (Biolegend, 1:75, clone BM8), CD11b-APC-Cy7 (BD Bioscience, 1:100, clone M1170) and Ly-6C-APC (Ebioscience; 1:75, clone 4K1.4). To quantify the level of ADAMTS1 in macrophages by flow cytometry, cells were co-stained with ADAMTS1 antibody (R&D Systems, 1:50, clone CDSL0115031). To quantify ADAMTS1 levels in macrophages after muscle injury, injured muscles were harvested day 1 post injury. Cells were analyzed and purified by FACS using a BD FACSAria II.
To confirm the co-localization of ADAMTS1 with macrophages by histology, cryosections of TA muscles were stained with F4/80-FITC (Biolegend, 1:75, clone BM8) and ADAMTS1 antibody (1:500 Santa Cruz Biotech, 1:500, clone A19) at 4 °C overnight. The sections were washed and incubated with fluorescent-dye conjugated secondary antibodies (1:1000, Invitrogen) for 30 min at room temperature. The sections were covered with antifade mounting medium (Vector Laboratories) and imaged.

Isolated liver NPCs were incubated with1 μl of anti-mouse FcγRII/III (Becton Dickinson, Franklin Lakes, NJ) to minimize non-specific antibody binding. The cells were then stained with anti-mouse CD45-V655 (eBioscience, 15520837), F4/80-APC/Cy7 (Biolegend, 123118), Ly6C-APC (BD Pharmingen, 560595), Ly6G-V450 (BD Pharmingen, 560603), CD146-PerCP-Cy5.5 (BD Pharmingen, 562134), CD44-PE (BD Pharmingen, 553134), anti-His-FITC (abcam, ab1206). In some experiments, cells were incubated with 2 μg rmChi3l1 for 2 hr before antibody staining. The cells were analyzed on a CytoFLEX LX Flow Cytometer (Beckman Coulter, Indianapolis, IN) using FlowJo software (Tree Star, Ashland, OR). For flow cytometric analysis, CD45⁺ cells were gated to exclude endothelial cells, hepatic stellate cells, and residue hepatocytes. Within CD45⁺ cells, CD44⁺ cells that bind to Chi3l1 were back-gated to determine the cells types.

To analyze immune cells (innate and adaptive) in the spleen, bone marrow or inguinal lymph nodes of 6- and 12-month-old Mlkl^WT/WT, Mlkl^–/–, Ripk3^WT/WT, and Ripk3^–/– mice, single-cell suspensions were subjected to osmotic red blood cell lysis and incubated with a combination of antibodies: CD4-BV421 (BD Biosciences #562891), CD8-PeCy7 (BD Biosciences #561097), CD19-PerCPCy5.5 (BD Biosciences #551001), CD11b-BV510 (BD Biosciences #562950) or CD11b-BV786 (BD Biosciences #740861), CD45-Alexa700 (BD Biosciences #560566), Ly6G-V450 (BD Biosciences #560603) and Ly6C-APCCy7 (BD Biosciences #560596). Samples were analyzed on an Aurora Cytex flow cytometer, with the automated volume calculator used to quantify absolute cell numbers. FlowJo v10.8 was used for all analyses.

Isolated brain tumour tissue was digested in cysteine-activated papain (Worthington, 50 U ml⁻¹ in HBSS, pH 7) with 300 U ml⁻¹ DNAse for 5 min at 37 °C, triturated, and strained through a 70 µm strainer. Cell suspension was resuspended in HBSS and subsequently underlayed with 35 and 70% Percoll (GE Healthcare) and centrifuged for 15 min at 800 × g. Interphase between layers was collected and washed using PBS with 1% bovine serum albumin and 0.1% NaN₃. After Fc receptor blocking (TruStain FcX™ (Clone 93) #101320, BioLegend), cells were incubated with fluorochrome-conjugated primary antibodies at 1:100 dilution. Antibodies for flow cytometry (V450-Ly6G, #560603; PE-Cy7-Ly6C #560593; PerCP-Cy™5.5-CD11b #550993; and APC-CD45 #559864) were purchased from BD Biosciences. The gating strategy is depicted in Supplementary Fig. 1: we selected CD11b^hiCD45⁺ population and separated Ly6G⁺ neutrophils and Ly6C⁺ newly infiltrated monocytes. The CD45^lowLy6C^low population is microglial cells and the CD45^hiLy6C^low population is cells of myeloid origin, differentiated TAMs. Analysis was performed using CytoFLEX platform (Beckman Coulter) with CytExpert software. Analysis of data was performed using FlowJo software. Fluorescence-activated cell sorting was performed using Sony SH800 cell sorter to collect GFP⁺ and GFP⁻ populations from NeuroD2:SmoA1;Atoh1:GFP tumours.

The anti‐α‐tubulin antibody, aphidicolin, and nocodazole were purchased from Sigma‐Aldrich. The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz. Antibodies against ATM, Flag, mouse cGAS, human cGAS, STING, MRE11, PARP1, H2A, H2A.X, γ‐H2A.X p‐IRF3, and IRF3 were from Cell Signaling Technology; Alexa 488‐anti‐Sca‐1 was from Invitrogen and PECY7‐anti‐cKit; V450‐Ly6G and FITC‐anti‐GR1 were from BD Pharmingen; 2′,3′‐cGAMP and immunostimulatory DNA (ISD) were from InvivoGen; and ATP was from New England Biology, while GTP, Rad51, and Lamin B1 antibody were from Abcam.