Anti mouse pe labeled antibody against ly6g

For flow cytometry analysis, mice were euthanized (i.p. 120 mg/kg ketamine, 12 mg/kg xylazine, and 0.08 mg/kg atropine), blood samples were collected, and retrograde perfusion of hearts was performed using ice‐cold PBS with 1% fetal bovine serum (FBS). Ventricles were dissected, rinsed in ice‐cold PBS, and minced with Medimachine (BD Biosciences Pharmingen) in PBS with 1% FBS. Tissue suspensions were filtered through a 70‐μm filter (BD Biosciences Pharmingen) and then centrifuged at 300g for 10 minutes at 4 °C. For staining, primary antibodies were added to the cells in a volume of 100 μL/10⁶ cells and incubated for 45 minutes on ice in the dark. After incubation, the cells were washed and resuspended in 300 μL of PBS with 1% FBS. Then, the blood samples were treated with red blood cell lysis buffer. The data were collected on a FACS Caliber Flow cytometer (BD FACS Calibur, USA) and analyzed using Flow Jo 10.1 software (Tree Star). For fluorescence detection, blood neutrophils were incubated with an anti‐mouse PE‐labeled antibody against Ly6G (eBioscience) and a Percp‐Cy5.5‐labeled antibody against CD11b (eBioscience); heart neutrophils were incubated with an anti‐mouse PE‐labeled antibody against Ly6G (eBioscience) and an FITC‐labeled antibody against CD45 (eBioscience).