To investigate leukocyte adhesion, we used a human flow chamber system. In brief, glass capillaries were coated with P-selectin (20 µg/ml; R&D Systems) and VCAM-1 (20 µg/ml; R&D Systems) or P-selectin (20 µg/ml; R&D Systems), VCAM-1 (20 µg/ml; R&D Systems) and SDF1 (20 µg/ml; R&D Systems) for 2 h. Chambers were blocked with 1% casein (Thermo Fisher Scientific) for 1 h and afterward perfused with isolated CD4 cells at a constant shear stress of 1 dyne/cm2. To investigate the capturing of CD4 cells, flow chambers were coated with P-selectin (20 µg/ml) alone and were perfused with isolated CD4 cells at a constant shear stress of 1 dyne/cm2 for 2 min and, subsequently, the number of rolling cells per field of view was determined. Videos were obtained with an inverted TS100 transmission light microscope (Nikon) equipped with a 10×/0.25 NA objective and a digital camera (Pixelfly; Cooke Corporation).
P selectin
Manufactured by R&D Systems
Sourced in United States
P-selectin is a cell adhesion molecule that is involved in the initial rolling and tethering of leukocytes to the endothelium during the inflammatory response. It is expressed on the surface of activated endothelial cells and platelets.
Automatically generated - may contain errors
Lab products found in correlation
E selectin, by R&D Systems (10 mentions)
Icam 1, by R&D Systems (7 mentions)
Casein, by Thermo Fisher Scientific (3 mentions)
Vcam 1, by R&D Systems (3 mentions)
Mcp 1, by R&D Systems (3 mentions)
P selectin, by Merck Group (3 mentions)
Icam 1, by Merck Group (2 mentions)
Human igg fc chimeric proteins, by R&D Systems (2 mentions)
Anti human igg fc pe, by Thermo Fisher Scientific (2 mentions)
Sdf 1, by R&D Systems (1 mentions)
Pe50 tubing, by BD (1 mentions)
Substrate reagent pack, by R&D Systems (1 mentions)
Autoanalyser, by Hitachi (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Aspirin, by Merck Group (1 mentions)
Clopidogrel, by Merck Group (1 mentions)
Hematrue hematology bench top analyzer, by Heska (1 mentions)
Il 1α, by Thermo Fisher Scientific (1 mentions)
Rhe selectin, by R&D Systems (1 mentions)
Icam 1, by Thermo Fisher Scientific (1 mentions)
36 protocols using p selectin
1
Leukocyte Adhesion Dynamics Under Flow
2
Investigating Murine Neutrophil Rolling Velocity
3
Selectin-Sialyl Lewisx Binding Assay
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Recombinant human L-selectin (10 ng/ml), P-selectin (10 ng/ml) or E-selectin (30 ng/ml) Fc chimeras (R&D Systems) were mixed with increasing concentrations of heparin or heparin derivatives in PBS before addition to 96-well ELISA plates precoated with BSA-Sialyl Lewisx (110 ng/well, R&D Systems) for 45 minutes at room temperature. The plates were washed three times with 200 μl of Quantikine wash buffer (R&D Systems) and the bound selectins were detected using an HRP-anti Fc antibody and Substrate Reagent Pack (R&D Systems) according to the manufacturer's instructions.
4
Vascular Biomarkers in Metabolic Profiling
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TC, HDL-C, TG, and Fasting plasma glucose concentration (FPG) were measured by the autoanalyser (HITACHI, Tokyo, Japan) with commercially available kits (Randox Laboratories, Crumlin, UK). Serum sPLA2-IIa protein was determined using a sandwich-type enzyme-linked immunosorbent assay (ELISA) kit (Cayman Chemicals, Ann Arbor, MI, USA). Serum sPLA2 activity was measured by a selective fluorometric assay (Cayman Chemicals) by using diheptanoyl thio-phosphocholine as fluorescent substrate. We used a solution of 0.1U bee venom PLA2 as a positive control to hydrolyze fluorescent substrate completely. The hydrolysis of substrate in the absence of serum sample was used as negative control. The intra-assay coefficient of variation was 4.2%. ELISAs were used to determine VCAM-1, ICAM-1, E-selectin, and P-selectin levels (R & D Systems, Minneapolis, MNUSA). All measurements were performed according to the manufacturers’ instructions. Each sample was tested in duplicate, and the means of the duplicates were used for the statistical analysis.
5
Immobilization of P-selectin and ICAM-1
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Dry powder of P-selectin and ICAM-1 were dissolved in Hank’s balanced salt solution (HBSS, Gibco, NY, USA). A coating region (5 mm × 5 mm) was marked in the center of each cover slide (Fisher Scientific Pittsburgh, PA, USA) with clean silicon rubber. Then, 40 μl of 500 ng/ml P-selectin (R&D, MN, USA) alone or plus 500 ng/ml ICAM-1 was directly adsorbed to the petri dish to incubate at 4°C for 12 h. After removing the excess molecules, the petri dishes were washed with PBS containing 2% BSA three times and incubated in 2% BSA in PBS for 2 h at room temperature to block nonspecific bindings. This P-selectin density of 500 ng/ml could support stable rolling (42 (link)).
6
Platelet-Monocyte Crosstalk in COVID-19
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To examine whether platelets from COVID-19 patients modulate TF expression in monocytes from healthy volunteers, purified platelets and monocytes were incubated ex vivo at 37°C in a 5% CO2 atmosphere. Each experimental point contained 2 × 105 monocytes from a healthy volunteer and 2 × 107 platelets from a COVID-19 patient or a heterologous healthy volunteer. In selected experiments, platelet-monocyte interactions were performed in the presence of neutralizing antibodies against P-selectin (R&D Systems) or isotype-matched control (20 µg/mL), or with the clinically approved anti-integrin αIIbβ3 monoclonal antibody abciximab (50 µg/mL), aspirin (100 µM; Sigma-Aldrich), or clopidogrel (300 µM; Sigma-Aldrich). After 0.5, 2, or 18 hours of interaction, cells were fixed with 4% paraformaldehyde and monocytes evaluated through flow cytometry as described previously in “Materials and methods.” The experiment was repeated using monocytes from 2 to 4 independent healthy volunteers with similar results, and representative data from 1 of the donors is shown.
7
Platelet and Inflammatory Marker Measurement
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Peripheral blood was collected via cardiac puncture 30 min after APE challenge using a 1-ml heparinized syringe. A total of 100 μl aliquots were immediately sampled and analyzed using a veterinary Hematrue hematology bench top analyzer (Heska Corporation Loveland, Colorado, U.S.A.). The number of platelets was measured. Commercial ELISA kits were used to determine CTS (CUSABIO, Cedarlane, Burlington, Canada), P-selectin, E-selectin, MCP-1, (R&D Systems, Minneapolis, MN, U.S.A.), MPO and VWF levels (Abcam, Shanghai, China).
8
Selectin-Mediated Cancer Cell Adhesion
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The adhesion of UTSCC 2, UTSCC 24A, UTSCC 24B, and Carey 24 cells on rhE-selectin and P-selectin (R&D Systems) and stimulated (10 ng/mL IL-1α, PeproTech, Hamburg, Germany) HUVECs was analyzed under physiological flow conditions in ibidiTreat µ-slide IV0.4 flow chambers, as previously described [51 (link)]. The applied shear rate was 0.5 dyn/cm2. Stimulated HUVECs were incubated with an adhesion blocking E-selectin mAb (BioLegend, San Diego, CA, USA) prior to the experiment [51 (link)]. Furthermore, cancer cells were pretreated with pronase, as described in the section above.
9
Functionalization of Culture Dishes for Cell Adhesion
10
Atherogenesis in ApoE-deficient Mice
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Eight-week-old male ApoE–/–/P–/–, ApoE–/–/PSGL1–/–, or ApoE–/– mice were fed a western diet (21% fat + 0.15% cholesterol). At the end of 12 weeks of treatment, the mice were euthanized and the brachiocephalic trunks and blood samples were collected for analysis. For splenic DC stimulation, wild-type (WT), PSGL1–/–, TLR4–/–, and MyD88–/– mice were subcutaneously (s.c.) infused with P-selectin at 5 mg/kg (R&D Systems, Minneapolis, MN, USA) for 1 day. Mice were s.c. infused with normal saline as a negative control group or treated with liposaccharide (LPS; 1 mg/kg; Sigma-Aldrich Corporation, St. Louis, MO, USA) as a positive control group. Three days later, the mice were killed and the spleens were collected. Single-cell suspensions were prepared and then analyzed by flow cytometry.
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