Alexa fluor 488 conjugated anti f4 80 antibody

The magnetically separated muscle-derived CD45⁺ cells were stained with a combination of Alexa Fluor 488-conjugated anti-F4/80 antibody (MF48020, Invitrogen, Carlsbad, USA) and Alexa Fluor 647 conjugated anti-Ly6G/Ly6C (GR-1) antibodies (108418, BioLegend, San Diego, USA) at room temperature for 15 minutes. Cells were gated based on their forward- and side-scatter characteristics. Macrophages were gated as GR-1 negative and F4/80 positive, while neutrophils as F4/80-negative and GR-1-positive cells. F4/80-positive macrophages were also analyzed for Ly6C, CD206 or MHCII expressions following staining with Ly6C PerCP-Cy5.5 (128012, BioLegend, San Diego, USA), CD206-PE (141705, BioLegend, San Diego, USA) or MHCII-FITC (107605, BioLegend, San Diego, USA) antibodies, respectively. Fluorescent intensity was detected with a Becton Dickinson FACSCalibur instrument.

Phagocytosis assay was performed as described previously [32 (link)]. Briefly, target C2C12 cell necrosis was induced by heating the cells for 10 minutes at 65 °C. C2C12 cells were stained with 1 µM CellTracker Deep Red Dye (ThermoFisher, Waltham, USA) and added to MΦs at 5:1 ratio (dead cell/MΦ). After 1-h co-culture, target cells were washed away extensively and MΦs were detached by EDTA. MΦs were labeled with Alexa Fluor 488-conjugated anti-F4/80 antibody (Invitrogen, Carlsbad, USA) for 20 min and the percentage of engulfing cells was determined on a Becton Dickinson FACSCalibur flow cytometer.

The magnetically separated muscle-derived CD45⁺ cells were stained with a combination of Alexa Fluor 488-conjugated anti-F4/80 antibody (MF48020, Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 647-conjugated anti-Ly6G/Ly6C (GR-1) antibodies (108418, BioLegend, San Diego, CA, USA) at room temperature for 15 min. The cells were gated based on their forward and side scatter characteristics. Macrophages were gated as GR-1^– and F4/80⁺, while neutrophils as F4/80^– and GR-1⁺ cells. This gating strategy was presented in our previous paper [12 (link)]. F4/80⁺ macrophages were also analyzed for expression of Ly6C, CD206, or major histocompatibility complex (MHC)II, following staining with the corresponding antibodies, Ly6C PerCP-Cy5.5 (128012, BioLegend, San Diego, CA, USA), CD206-PE (141705, BioLegend, San Diego, CA, USA), or MHCII-FITC (107605, BioLegend, San Diego, CA, USA), respectively. Fluorescent intensity was detected with a Becton Dickinson FACSCalibur instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, USA).