Journal: bioRxiv

Article Title: An Unexpected Role of Neutrophils in Clearing Apoptotic Hepatocytes In Vivo

doi: 10.1101/2023.02.08.527616

Figure Lengend Snippet: (A) Intravital microscopy images of liver tissues from WT and MRP8cre/DTR mice. Neutrophils are labeled with an i.v. injection of anti-Ly6G antibody (green), KCs are labeled with anti-F4/80 antibody (red), and apoptotic cells are labeled with Annexin V (blue). Neutrophils inside and KCs associated with apoptotic hepatocytes are detected and analyzed with IMARIS software as described in Methods. The distances from neutrophils to the apoptotic hepatocyte border are recorded in table S6. A total of 24 apoptotic cells were observed in the WT liver with an average of 2 burrowed neutrophils. Scale bar, 100 μm. (B) Intravital image sequences of neutrophils phagocytosing apoptotic hepatocytes in mouse livers at indicated time points. Neutrophils are labeled with an i.v. injection of anti-Ly6G antibody (green), and apoptotic cells are labeled with Annexin V (red). A total of 13 apoptotic cells with burrowed neutrophils were observed in 12 WT mouse livers. Scale bar, 20 μm. (C) Electron microscopy images of apoptotic mouse hepatocytes occupied by neutrophils. The apoptotic hepatic nucleus (AN) is evident by distorted nuclear membrane (pointed by black arrowheads). The neutrophils are indicated by white arrowheads with a characteristic multilobed nucleus. 29 apoptotic cells with burrowed neutrophils were observed. Scale bar, 5 μm. Data are representative of three independent experiments.

Article Snippet: For antibody-based depletion, wild-type mice were injected i.p. with anti-mouse Ly6G clone 1A8 (Bio X Cell) supernatant containing 0.5 mg protein every 48 h for 1 month.

Techniques: Intravital Microscopy, Labeling, Injection, Software, Electron Microscopy, Membrane

Journal: Experimental & molecular medicine

Article Title: Neutrophil extracellular traps mediate the crosstalk between plaque microenvironment and unstable carotid plaque formation.

doi: 10.1038/s12276-024-01281-4

Figure Lengend Snippet: Fig. 4 MD-1 induces the formation of NETs. a Isolation and validation of neutrophils (Giemsa staining, Trypan blue staining, and cell counter, scale bar = 20 μm). b Hochest staining and ELISA were used to quantify the concentration and time course of MD-1-induced NETs. c NETs were quantified in the negative control group, the MD-1 (75 ng/ml) group, and the two positive control groups. d Co-IP revealed that MD-1 and TLR4 coexisted on neutrophils. e The interaction between MD-1 and TLR4 in peripheral blood neutrophils from carotid plaque patients and peripheral blood neutrophils from healthy volunteers induced under different conditions was analyzed by Duolink PLA (scale bar = 100 μm). f Western blot analysis showed that neutrophils induced with MD-1 upregulated TLR4 expression and increased the phosphorylation of IRAK1/IRAK4, p38 MAPK, and ERK1/2 in a time-dependent manner (from 0 to 60 min). t total value; p phosphorylation value. β-Actin was used as an internal control.

Article Snippet: We constructed a neutrophil depletion model based on the in vivo injection of InVivoPlus anti-mouse Ly6G (BP0075-1-5MG, BioXCell, New Hampshire, USA).

Techniques: Isolation, Biomarker Discovery, Staining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control, Positive Control, Co-Immunoprecipitation Assay, Western Blot, Expressing, Phospho-proteomics, Control