Efferocytosis assay kit

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).

An efferocytosis assay kit (Cayman, 601770) was used following the manufacturer’s instructions. Neutrophils were labelled with CFSE and cultured in RPMI with 2% serum for 12 h to induce cell death. Bone marrow-derived macrophages cultured for 7 days were seeded at a density of 4 × 10⁵ cells per well in a 6-well plate with DMEM/F12 containing 10% FBS and 100 units ml⁻¹ penicillin/streptomycin. Prior to the assay, macrophages were pre-treated with CGRP (1 or 20 nM) for 24 h. Macrophages were collected, labelled with CytoTell Blue, and then incubated with CFSE-labelled dead/dying neutrophils at different ratios (1:1, 1:2, and 1:4) at 37 °C for 15 min. The reaction was stopped by washing cells with ice-cold PBS containing 5% FBS and 1 mM EDTA. Cells were analysed with BD LSR Fortessa X-20 and FlowJo software (BD Biosciences). Macrophages were identified by CytoTell Blue-positive staining, and the efferocytosis index was calculated as the percentage of CFSE-positive cells in CytoTell Blue-labelled macrophages.