Purified anti-human CD62P (P-Selectin) 304902 from BioLegend

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Nature communications
March 21 2019

Fig 1: HIT IgG induces NETosis via activated platelets and by direct neutrophil activation. a Platelets and neutrophils were resuspended in autologous plasma at a 20:1 ratio and incubated with KKO plus heparin at 37 °C for 1 h, platelets were stained with anti-CD41 Alexa 647 (red), neutrophils with Hoechst (blue), extracellular DNA with Sytox green (green), and CitH3 with anti-CitH3 Alexa 594 (magenta) and then perfused on VWF-coated microchannels at a shear stress of 67 dynes/cm2 at 37 °C for up to 20 min. Samples were fixed at the end of the experiment. Panels show confocal images of a representative thrombus of three individual experiments, using different donors imaged with a 63x oil immersion objective. Scale bar: 20 μm. b Purified neutrophils were supplemented with HIT complex-activated platelets in the absence or presence of anti-CD62p or anti-CD162 and incubated at 37 °C. DNA release was monitored by confocal microscopy for over 5 h by Sytox green fluorescence. Total DNA, Hoechst staining. The graph shows the fluorescence intensity ratio of extracellular DNA/total DNA vs. time (n = 3). c Purified neutrophils were resuspended in autologous plasma and incubated with KKO plus heparin at 37 °C for 1.5 h. Neutrophils were stained with anti-CD15 Alexa 647 (yellow), DNA with Hoechst (blue), the extracellular DNA with Sytox green (green) and CitH3 with anti-CitH3 Alexa 594 (magenta) and perfused on P-selectin-coated microchannels at a shear stress of 67 dynes/cm2 at 37 °C for up to 30 min. Samples were fixed at the end of the experiment. The panels show confocal images of a representative thrombus of three individual experiments, using different donors imaged with a ×40 water immersion objective. Scale bar: 20μm. d Percentage area coverage versus time of P-selectin-coated surfaces perfused with purified neutrophils in the presence of HIT IgG, normal IgG or HIT IgG plus IV.3 antibody (all in the presence of heparin). DNA (left panel), neutrophils (right panel). n = 3; Mean ± s.d. is shown. Statistics: one-way ANOVA with Tukey’s correction for multiple comparisons. **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data for (b, d) are provided as a Source Data file
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Nature communications
March 21 2019

Fig 2: NETosis is required for thrombus formation in HIT. a Neutrophils plus platelets were treated with PF4, heparin and normal IgG (n = 4, orange), or HIT IgG plus heparin without (n = 3, red) or with GSK484 (n = 5, green) or IV.3 antibody (n = 4, magenta). Treated neutrophils alone are also shown (n = 4, blue). The extracellular DNA release determined as in Fig. 4b. Mean ± s.e.m. b Quantification of neutrophil–platelet aggregates, mean ± s.e.m. c NETs after 5 h treatment with antibodies and inhibitors, mean ± s.e.m. Fluorescent microscopy images of thrombi in microchannels. d Blood pre-treated with KKO plus heparin plus vehicle control, DNase I or IV.3. Neutrophils (anti-CD15 AF594, left panel, blue), nucleated cells (Hoechst, middle panel, blue), the extracellular DNA (Sytox green, green) and platelets (anti-CD41 AF647, magenta). Scale bar: 50 µm. e Area coverage of neutrophils, DNA and platelets. n = 3, mean ± s.d. f Whole blood (WB) (left panels), neutrophil (Neut) depleted blood (middle panels) or depleted blood reconstituted (recons) with autologous neutrophils (right panels) incubated with KKO and heparin, treated as described in (d). Plt, platelets; Scale bar: 50 µm. g Quantification as in (e), n = 3, mean ± s.d. h Fc?RIIa+/hPF4+ mice treated with normal (n = 5, black) or HIT IgG plus heparin plus vehicle control (n = 7, dotted red) or anti-CD62p (n = 3, green), agIV.3 antibody (n = 8, blue), GSK484 (n = 8, magenta) or DNase I (n = 8, grey). Mean ± s.e.m. i Box-and-whiskers plot of cfDNA in mouse plasma described in (h) (n = 4 for normal IgG, anti-CD62p; n = 5 for DNase I; n = 6 for vehicle, IV.3, GSK484). Middle line, bounds of box and whiskers represent the median, 25th to 75th percentiles, and minimum and maximum values, respectively. j Mouse lungs imaged as described in (3b) and plot of fluorescence intensity. Mean ± s.e.m. k Representative western blots of mouse plasma described in (h) probed with anti-CitH3 antibody. Arrowhead, CitH3. Arrow, transferrin (transf, loading control). Statistics: (b, c, h, i, j) Kruskal–Wallis test for comparison of groups with versus without inhibitor. P-values adjusted relative to HIT IgG (vehicle). e, g one-way ANOVA with Tukey’s correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Source data for (a, b, c, e, g, h, i, j, k) are provided as a Source Data file

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Purified anti-human CD62P (P-Selectin)

BioLegend's Purified anti-human CD62P (P-Selectin) is a Mouse monoclonal antibody. The Purified anti-human CD62P (P-Selectin) Antibody was generated using Selectin P, and Selp as the antigen. It reacts with Homo Sapiens, and Human. This antibody has been shown to work in applications such as: Flow Cytometry, Immunohistochemistry, and Immunohistochemistry - fixed.

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Purified anti-human CD62P (P-Selectin) [AK4]

Nature communications

Nature communications

Purified anti-human CD62P (P-Selectin) from BioLegend

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BioLegend

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Purified anti-human CD62P (P-Selectin) from BioLegend

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