Human Siglec-5/Siglec-14 Antibody (194128)

Detection of Siglec-5/Siglec-14 by Flow Cytometry

Siglec-5 is required for mediating the interaction of human monocytes and endothelial cells.(A) Before adding THP1-RFP cells, the activated HUVECs were pretreated with or without 1.5 μg/ml of anti-siglec-4a or anti-Siglec-5. Then THP1-RFP cell adherent was analyzed by the same procedure described above.(B) 6 random fields per well were imaged from (A) and the cell numbers in each field were counted by Qupath. The adherent cell numbers were averaged and presented as mean ± SD, n = 6. One-way ANOVA analysis followed by Tukey’s test was performed. **P < 0.01, ***P < 0.001, ****P < 0.0001.(C) Western blot of cell lysates from HUVECs treated with TNF alpha (10 ng/ml) and LPS (1 μg/ml) for different time points as indicated. The blot was probed by primary antibodies as indicated and imaged by secondary antibody-HRP.(D) HUVECs were stimulated with TNF alpha and LPS overnight and then incubated with anti-Siglec-5 and Alex468-tagged anti-IgG secondary antibody complex. THP1 cells were also stained as a control. The stained cells were analyzed by Flow cytometry. Image collected and cropped by CiteAb from the following open publication (https://linkinghub.elsevier.com/retrieve/pii/S0167488925001223), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Siglec-5/Siglec-14 by Immunocytochemistry/ Immunofluorescence

Siglec-5 is required for mediating the interaction of human monocytes and endothelial cells.(A) Before adding THP1-RFP cells, the activated HUVECs were pretreated with or without 1.5 μg/ml of anti-siglec-4a or anti-Siglec-5. Then THP1-RFP cell adherent was analyzed by the same procedure described above.(B) 6 random fields per well were imaged from (A) and the cell numbers in each field were counted by Qupath. The adherent cell numbers were averaged and presented as mean ± SD, n = 6. One-way ANOVA analysis followed by Tukey’s test was performed. **P < 0.01, ***P < 0.001, ****P < 0.0001.(C) Western blot of cell lysates from HUVECs treated with TNF alpha (10 ng/ml) and LPS (1 μg/ml) for different time points as indicated. The blot was probed by primary antibodies as indicated and imaged by secondary antibody-HRP.(D) HUVECs were stimulated with TNF alpha and LPS overnight and then incubated with anti-Siglec-5 and Alex468-tagged anti-IgG secondary antibody complex. THP1 cells were also stained as a control. The stained cells were analyzed by Flow cytometry. Image collected and cropped by CiteAb from the following open publication (https://linkinghub.elsevier.com/retrieve/pii/S0167488925001223), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Siglec-5/Siglec-14 by Flow Cytometry

Identification of Siglec-5 as the binding partner of glycoRNAs from THP1 cells.(A) FACS analysis of THP1 cells labeled by pre-complex of E-selectin-Fc, P-selectin-Fc, VCAM-1-Fc, ICAM-1-Fc, Siglec-4a-Fc, Siglec-5-Fc and anti-Fc-FITC (left). The images taken from these cells by microscopy (right).(B) FACS analysis of THP1 cells pretreated with or without RNase then stained with the indicated pre-complex.(C) The images taken from these cells in (B) by microscopy. The fluorescence intensity from biological triplicate was analyzed by Cytation 3 Cell Imaging Multi-mode Reader (Biotek Instruments) and presented as mean ± SD, n = 6. One-way ANOVA was performed. **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s means no significance.(D) Biotinylated RNA was incubated with pre-binding anti-Fc-beads with indicated protein-Fc chimera and then analyzed by northern blot as described above.(E) Native RNAs were analyzed by northern blot. After blocking, the membrane was probed by pre-complex of Siglec-4a-Fc (1.5 μg/ml) or Siglec-5-Fc (1.5 μg/ml) with anti-Fc-HRP (1.5 μg/ml) for 2 h at 4 °C. The blot was imaged by iBright 1500. Blot of biotinylated RNA from labeled THP1 cells was probed by anti-biotin-HRP. Image collected and cropped by CiteAb from the following open publication (https://linkinghub.elsevier.com/retrieve/pii/S0167488925001223), licensed under a CC-BY license. Not internally tested by R&D Systems.