Anti ly6g bv421 antibody

Apoptosis was assessed morphologically, as reported previously by our group [8 (link),39 (link)]. Briefly, cells (5 × 10⁴) collected 8 h after LPS administration or 18 h after MSU injection were cyto-centrifuged, fixed, and stained with May-Grünwald-Giemsa and counted using oil immersion microscopy (×100 objective) to determine the proportion of cells with distinctive apoptotic morphology in a blind manner. This was assessed by recognizing cells presenting pyknosis—the nuclei are dense and compacted and have begun to break-up (a process named “karyorrhexis”) resulting in multiple spheres of dark-staining nuclear chromatin. At least 500 cells were counted/slide, and results are expressed as the mean ± SEM of percentage of cells with apoptotic morphology. Apoptosis was also evaluated by western blot, assessing the cleavage of caspase-3 and by flow cytometry. For in vivo experiments, cells were collected and surface-staining for 30 min with anti-LY6G-BV421 antibody (eBioscience) and then labelling with annexin-V APC and PI, and for in vitro experiments, isolated cells was labelled with annexin-V APC and PI as an index of loss of nuclear membrane integrity (PE Annexin V Apoptosis Detection Kit; BD PharmingenTM; San Jose, CA, USA).

Apoptosis and efferocytosis were assessed by flow cytometry, as described previously (Dalli et al., 2013 (link)). Mice were injected with MSU crystals and 12 h later they were treated locally with PI3K inhibitors. For the apoptosis assays, the lavage of the knee was performed 4 h after the treatment with drugs. Cells were surface-stained for 30 min with anti-LY6G-BV421 antibody (eBioscience) and then labeled with annexin-V FITC and PI, as an index of loss of nuclear membrane integrity (PE Annexin V Apoptosis Detection Kit; BD PharmingenTM; United States). For the efferocytosis assays, joint wasy was performed 6 h after the treatment with PI3K inhibitors and cells were surface-stained for 30 min with anti-F4/80-PECy7 antibody (eBioscience). Then, cells were fixed for 10 min, treated with 1× permeabilization wash (Cytofix/Cytoperm Kit; BD Biosciences) and intracellularly stained with anti-LY6G-BV421 antibody. Macrophage efferocytosis was assessed as a frequency of macrophages containing neutrophils (F4/80+ Ly6G+ cells).

Apoptosis was assessed morphologically, as reported previously (Barroso et al., 2017 (link)). In brief, 5 × 10⁴ cells collected 18 h after mBSA challenge were cyto-centrifuged, fixed, and stained with May–Grünwald–Giemsa and counted using oil immersion microscopy (×100 objective) to determine the proportion of cells with distinctive apoptotic morphology in a blind manner. Of note, cells were considered apoptotic when they exhibited chromatin condensation, nuclear fragmentation, and formation of apoptotic bodies outside or inside of macrophages (Poon et al., 2014 (link)). At least 500 cells were counted/slide, and results are expressed as mean ± SEM of percentage of cells with apoptotic morphology. Apoptosis was also evaluated by flow cytometry (FACS Canto II, BD Biosciences). Then, mice were injected with mBSA, and 12 h later, locally treatment with BCA was performed. For the apoptosis assays, the lavage of the knee was performed 4 h after the treatment with drugs. Cells were surface-stained for 30 min with the anti-LY6G-BV421 antibody (eBioscience) and then labeled with annexin-V-APC and propidium iodide (PI), as an index of loss of nuclear membrane integrity (PE Annexin V Apoptosis Detection Kit; BD PharmingenTM; United States).