Journal: Frontiers in Immunology

Article Title: Identification of Extracellular Actin As a Ligand for Triggering Receptor Expressed on Myeloid Cells-1 Signaling

doi: 10.3389/fimmu.2017.00917

Figure Lengend Snippet: FACS analysis of surface triggering receptor expressed on myeloid cells-1 (TREM-1) and its ligand on cells. Mice ( n = 5) were inoculated with LPS for 6 h or mock treated with PBS as a control. The blood cells were collected, and red blood cells were removed for analysis of TREM-1 expression or analysis of the distribution of TREM-1-interacting proteins. All experiments were done in triplicate. (A) FACS analysis of TREM-1 expression on the cell surface with phycoerythrin (PE)-conjugated rat anti-mouse TREM-1 or PE Rat IgG2a, κ Isotype ctrl Antibody, allophycocyanin-conjugated anti-mouse F4/80, and Percp/cy5.5-conjugated anti-mouse Ly-6G. The signal for TREM-1 was specific (Figure in Supplementary Material), and the cells expressing TREM-1 was further analyzed (Figure in Supplementary Material). (B) FACS analysis of the distribution of TREM-1-interacting proteins on cells with Cy5.5-NHS-Ester-labeled recombinant extracellular domain of mouse TREM-1 (rTREM-1), PE/Cy7-conjugated anti-mouse CD41, and PE-conjugated anti-mouse Ly-6G. The cells expressing the ligand for TREM-1 were shown in details (Figure in Supplementary Material).

Article Snippet: The cells were then stained with allophycocyanin-conjugated anti-mouse F4/80 (BioLegend), Percp/cy5.5-conjugated anti-mouse Ly-6G (BioLegend), and phycoerythrin (PE)-conjugated rat IgG2A anti-mouse TREM-1 (R&D systems) or PE-conjugated Rat IgG2a, κIsotype Ctrl Antibody (BioLegend) to analyze which cells expressed surface TREM-1.

Techniques: Control, Expressing, Labeling, Recombinant

Journal: Frontiers in Immunology

Article Title: Identification of Extracellular Actin As a Ligand for Triggering Receptor Expressed on Myeloid Cells-1 Signaling

doi: 10.3389/fimmu.2017.00917

Figure Lengend Snippet: Analysis of the co-localization of triggering receptor expressed on myeloid cells-1 (TREM-1) and actin by laser scanning confocal microscopy. RAW264.7 cells were mock treated with PBS or treated with LPS, platelets, or rACTIN. All cells were fixed and then incubated with mouse TREM-1 antibody antigen affinity-purified polyclonal goat IgG and rabbit anti-beta actin polyclonal antibody, followed by FITC-conjugated affinipure donkey anti-rabbit IgG (H + L) and CY3-conjugated affinipure donkey anti-goat IgG (H + L). After staining the nuclei with Hoechest 33258, the slides were analyzed with a LSM880 with Airyscan laser scanning confocal microscope and ZEN2.3 LITE software. The scale bars in the figure represent 10 µm.

Article Snippet: The cells were then stained with allophycocyanin-conjugated anti-mouse F4/80 (BioLegend), Percp/cy5.5-conjugated anti-mouse Ly-6G (BioLegend), and phycoerythrin (PE)-conjugated rat IgG2A anti-mouse TREM-1 (R&D systems) or PE-conjugated Rat IgG2a, κIsotype Ctrl Antibody (BioLegend) to analyze which cells expressed surface TREM-1.

Techniques: Confocal Microscopy, Incubation, Affinity Purification, Staining, Microscopy, Software

Journal: Frontiers in Immunology

Article Title: Identification of Extracellular Actin As a Ligand for Triggering Receptor Expressed on Myeloid Cells-1 Signaling

doi: 10.3389/fimmu.2017.00917

Figure Lengend Snippet: Analysis of the co-localization of triggering receptor expressed on myeloid cells-1 (TREM-1) and actin in lung sections of cecal ligation and puncture (CLP) mice or healthy control mice. The lung sections from CLP mice ( n = 3) or mock-treated mice ( n = 3) were stained with mouse TREM-1 antibody antigen affinity-purified polyclonal goat IgG and rabbit anti-beta actin polyclonal antibody, followed by FITC-conjugated affinipure donkey anti-rabbit IgG (H + L) and CY3-conjugated affinipure donkey anti-goat IgG (H + L). The cells were analyzed with Fluoview™ Fv1000 laser scanning confocal microscope and FV10-ASW3.1 viewer software. The scale bars in the figure represent 20 µm.

Article Snippet: The cells were then stained with allophycocyanin-conjugated anti-mouse F4/80 (BioLegend), Percp/cy5.5-conjugated anti-mouse Ly-6G (BioLegend), and phycoerythrin (PE)-conjugated rat IgG2A anti-mouse TREM-1 (R&D systems) or PE-conjugated Rat IgG2a, κIsotype Ctrl Antibody (BioLegend) to analyze which cells expressed surface TREM-1.

Techniques: Ligation, Control, Staining, Affinity Purification, Microscopy, Software