Skeletal muscle dissociation kit

Details of skeletal muscle dissection have been previously described (13 (link)). In brief, muscle was dissected from Acvr1^{[R206H]FlEx/+}; Gt(ROSA26)Sor^CreERT2/+ mice and dissociated using the Skeletal Muscle Dissociation Kit (Miltenyi Biotec) and gentleMACS Octo Dissociator with heaters (Miltenyi Biotec), in accordance with the manufacturer’s instructions. Following centrifugation at 300g and 4°C for 10 minutes, the supernatant was discarded, and the pellet was resuspended in growth media (Dulbecco’s modified Eagle medium (DMEM; Life Technologies) with 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco), and 16.6% fetal bovine serum (FBS; lot 192K18, Avantor). Cells were then plated onto tissue culture flasks (Corning). FACS was performed on single cells incubated with anti–mouse PDGFRA APC (clone APA5, eBioscience) to label FAPs, as previously described (13 (link), 35 (link)). FACS-isolated FAPs were seeded at a density of 2000 cells/cm² onto tissue culture flasks (Corning) in growth media and maintained at 37°C in a humidified atmosphere with 5% CO₂. Media were changed every other day. Prior to use, FAPs were treated with 2 μM (Z)-4-hydroxytamoxifen (Sigma-Aldrich) for 48 hours to induce inversion of the R206H-containing exon. All experiments were conducted with FAPs passaged fewer than 3 times.

Excised muscles were transferred into a plastic Petri dish with cold PBS and visible adipose and connective tissues were removed with a scalpel. Muscles were cut into small pieces using scissors and the shredded muscle pieces were treated using the Skeletal Muscle Dissociation Kit (Miltenyi Biotec, Calderara di Reno-Bologna, Italy) according to the manufacturer’s instructions in order to isolate satellite cells. Purity of the suspension was checked by flow cytometry by labeling cells with anti-alfa7 integrin-PE conjugated antibody (Miltenyi Biotech) and ranged from 88% to 99% (Supplementary Figure 2). The purified cells were then centrifuged, resuspended in DMEM supplemented with 10% FBS, 20% HS and 3% CEE, and seeded in collagen precoated 35 mm dishes at 37°C under 5% CO₂. After two days, medium was changed and cells were treated or not with TMZ.

Muscle tissue subjected to single cell isolation was harvested and stored on ice in serum-free Dulbecco’s Modified Eagle Medium (DMEM) immediately prior to processing. Biochemical and mechanical dissociation of hindlimb mouse tissues for isolation of muscle-infiltrating cells from recombinant HRS- versus PBS-immunized mice was performed using the Miltenyi Biotec Skeletal Muscle Dissociation Kit and a gentleMACS Octo-Dissociator (Miltenyi Biotec, Gaithersburg, Maryland). Following this dissociation step, single cell suspensions were sequentially filtered using 70 micron and 30 micron cell strainers to eliminate residual muscle fibers and washed with 1X PBS. Isolated cells were resuspended in appropriate volumes of PBS for cDNA library preparation and scRNAseq analysis or frozen in media containing 10% DMSO for future flow cytometry analysis.

GA muscles were dissociated into single-cell suspensions using a skeletal muscle dissociation kit (Miltenyi Biotec) according to the manufacturer's instructions. Mechanical disaggregation was performed via gentleMACS Dissociator using the m_muscle_01 program (Miltenyi Biotec).

For collection of the spleen for flow cytometry, the whole spleen was dissociated in a total of 10 mL PBS containing 2% fetal calf serum (FCS) using a GentleMACS Dissociator tissue homogenizer with matching C tubes (Miltenyi Biotec, Macquarie Park, Australia) on “spleen 3” setting, twice. For collection of muscle for flow cytometry, hamstrings were cut into 1 mm pieces, and up to 0.5 g of tissue was used per skeletal muscle dissociation kit as per manufacturer's instructions (Miltenyi Biotech, Germany). Bone marrow was isolated by flushing one femur with 1 mL PBS containing 2% FCS. Then, 0.5 to 1.0 mL of blood erythrocytes were harvested by terminal cardiac puncture under anesthesia, and blood erythrocytes were lysed as described.^{42 (link)}

Ovalbumin Alexa Fluor 647 conjugate (OVA-AF647) from Thermo Scientific (catalog # O34784) was used for cell recruitment studies. GentleMACS Dissociator from Miltenyi Biotec was used along with the Skeletal Muscle Dissociation Kit (130-098-305) to prepare single cell suspension of muscles. PBS and HBSS medium was obtained from GIBCO and Collagenase D and DNase I were procured from Roche. For flow cytometry, cells were stained with Live/Dead Fixable Yellow Dead Cell Stain kit (Invitrogen) and combinations of the following antibodies: α-Ly6C-FITC, α-Ly6G-PE, α-CD11b-PE-Cy7, α-CD3-PerCpCy5.5 (all from BD Pharmingen) and α-I-A/I-E-Alexa-Fluor700, α-F4/80-PacificBlue, α-CD11c-APC-eFluor780 (all from eBioscience). Mice were divided into the following experimental groups: naïve, vaccinated with non-adjuvanted OVA-AF647 antigen or OVA-AF647 mixed 1:1 v/v with SEA20, MFA160 or SEA160 respectively. Each mouse was administered 50 μl bilaterally into the quadriceps. Three mice per group were euthanized at 6 h and 72 h, and six mice per group were euthanized at 24 h and 48 h. The quadriceps muscles and dLN were harvested for all mice. Single cells were isolated from muscles using Skeletal Muscle Dissociation Kit according to the manufacturer’s instructions. Single cell isolation from dLN was done as described before^{7 (link)}.

Different cell types were purified from the heart 2 days after LAD ligation. Cardiomyocytes were isolated as described^{56 (link)}. For endothelial cells, inflammatory cells and fibroblasts, hearts were digested to single cell suspension using the Skeletal Muscle dissociation kit (Miltenyi Biotech). Endothelial cells were positively selected using anti-CD31 coated magnetic beads (Miltenyi Biotech), following manufacturer instructions. CD31-depleted cells were subsequently incubated with anti-CD45 magnetic beads (Miltenyi Biotech) to separate inflammatory cells from cardiac fibroblasts. Purified cells were pelleted at 4 °C at 300 × g for 5 min for RNA extraction.