After pro-inflammatory pretreatment and myoglobin (or control) treatment, the RAW264.7 cells were characterized as follows. The cells were measured for oxidative stress using the ROS-Glo H2O2 assay according to the manufacturer’s instructions (G8820, Promega). Luminescence was measured using a Tecan Spark M10 microplate reader (Tecan, Männedorf, Switzerland). The cells were also measured using an efferocytosis assay kit (Cayman Chemical, MI) according to the manufacturer’s instructions. Briefly, apoptotic bodies were created from staurosporine-treated C2C12 myoblasts (CRL-1772, ATCC) and labeled with CFSE fluorescence. Apoptotic bodies (or cell-free media control [CFM]) were added to wells for 16 hr prior to aspiration of the unengulfed apoptotic bodies, washing, and measurement. Absolute fluorescence was measured as integrated intensity for each well using a microplate reader (Tecan Spark M10, Männedorf, Switzerland) with excitation at 490 nm and emission at 525 nm.
RAW264.7 monocyte and C2C12 myoblast cell lines were authenticated using a commercial STR profiling service (Mouse Cell Authentication Service, ATCC 137‐XV) and tested negative for mycoplasma using the MycoAlert PLUS mycoplasma detection kit (LT07-705; Lonza, Basel, Switzerland).
RAW264.7 monocyte and C2C12 myoblast cell lines were authenticated using a commercial STR profiling service (Mouse Cell Authentication Service, ATCC 137‐XV) and tested negative for mycoplasma using the MycoAlert PLUS mycoplasma detection kit (LT07-705; Lonza, Basel, Switzerland).