Pe conjugated anti ly6c antibody hk1

TA muscles from CTX-injured animals were carefully isolated with fine scissors and fascia was removed^{29 (link)}. Despite alterations in the integrity of the muscle tissue during injury (e.g., swelling), the tissue remains intact during collection. Muscles were weighed and then placed directly into ice-cold PBS. Muscles were then dissociated in RPMI containing 0.2% collagenase B (Roche Diagnostics GmbH) at 37°C for 1 hour and filtered through a 100 μm and a 40 μm filter. CD45⁺ cells were isolated using magnetic sorting (Miltenyi Biotec). For FACS, macrophages were incubated with Fcγ receptor blocking antibodies and with 10% normal rat serum: normal mouse serum 1:1 mix, then stained with a combination of PE-conjugated anti-Ly6C antibody (HK1.4, eBioscience), APC-conjugated F4/80 antibody (BM8, eBioscience) and FITC-conjugated Ly6G antibody (1A8, Biolegend). Ly6C^hi F4/80^lo Ly6G^neg macrophages, Ly6C^lo F4/80^hi Ly6G^neg macrophages and Ly6G^hi Ly6C^int F4/80^neg neutrophils were quantified. In each experiment, samples were processed in parallel to minimize experimental variation. Cells were analyzed on a BD FACSAria III or MoFlo Astrios sorter (Summit v6 software) and data analysis was performed using BD FACSDIVA and FlowJo V10 software. Gating strategy is shown in Supplementary Fig. 2a–b.