Following treatment, for underflow, isolated human washed platelets were perfused over the HMVEC‐dLyAd at a wall shear rate of 50/s at 37°C for 8 minutes. PBS was then added to remove the nonadhered platelets. For static treatments, the HMVEC‐dLy‐Ad were treated with washed platelets for 1 hour. Cells were then fixed with paraformaldehyde 2% and immunofluorescence analysis was performed following incubation with anti‐CD61 (Clone: VI‐PL2, Biolegend).
Anti cd61
Manufactured by BioLegend
Anti-CD61 is a monoclonal antibody that binds to the CD61 antigen, which is also known as the platelet glycoprotein IIIa (GPIIIa) or integrin beta 3 (ITGB3). CD61 is expressed on the surface of platelets and is involved in platelet activation and aggregation.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd34, by BD (1 mentions)
Anti cd45, by BioLegend (1 mentions)
Annexin 5, by Abcam (1 mentions)
Anti cd133, by BioLegend (1 mentions)
Mitotracker deep red, by Thermo Fisher Scientific (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Annexin 5, by BioLegend (1 mentions)
Anti cd45, by Beckman Coulter (1 mentions)
Anti cd41, by Beckman Coulter (1 mentions)
4 protocols using anti cd61
1
Platelet Adhesion to Endothelial Cells
2
Phenotyping Megakaryocytes and Platelets
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The phenotype of differentiating MKs and PLTs was analyzed using FITC or PE conjugated monoclonal antibodies anti-CD34 (Becton Dickinson) and anti-CD61 (BioLegend). Cells were incubated with antibody (diluted 1:100 for 15 min), washed with PBS, and analyzed in a cytofluorimeter (Epics XL–MCL Coulter).
3
Characterization of Circulating Membrane Microparticles
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Membrane MP subpopulations of HFD‐ and normal‐fed mice were discriminated in PFP according the expression of membrane‐specific antigens by flow cytometer. Phenotype of endothelial MPs was performed using anti‐CD54 labeling, characterization of platelet, leukocyte, erythrocyte MPs, and MPs from progenitor cells was performed using anti‐CD61, anti‐CD45, anti‐Ter‐119/erythroid cell, and anti‐CD133 labeling, respectively, (BioLegend, San Diego, CA). Also, Annexin V (BioVision Inc., Mountain View, CA) binding was used to numerate phosphatidylserine‐expressing circulating MPs. Irrelevant mouse IgG was used as an isotype‐matched negative control for each sample.
4
Isolation and Characterization of Platelet Microparticles
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Peripheral blood was centrifuged at 1000g for 5 min. Plasma was then centrifuged at 1500g for 20 min to obtain platelet poor plasma (PPP). 1 mL of PPP was centrifuged for 30 min at 20,000g to pellet microparticles (MPs). The MP pellet washed once in 500 µL calcium-free HBS complemented with 0.2% bovine serum albumin (pH 7.3) by 45 min centrifugation at 20,000g. MPs were labeled with MitoTracker Deep Red (200 nM; Invitrogen, Breda, The Netherlands) in HBS at room temperature containing calcium and subsequently stained with anti-CD61, anti-CD62p, Annexin-V (all Biolegend), anti-CD45 and anti-CD41 (both Beckman Coulter). MPs were analyzed using a Cytoflex flow cytometer (Beckman Coulter) including the sensitive violet Side Scatter (405 nM; VSSC) and FSC for detection of ultrasmall particles (1 µm)54 (link). Platelet MPs (PMPs) were selected based on aforementioned markers. Counting beads (Sphero Nano fluorescent, Spherotech, Fulda, Germany) of different sizes were included for reference to correct for concentration and size. All solutions for PMP isolation and staining were centrifuged at 20,000g for 20 min to remove (fluorescent) aggregates. A gating strategy is provided in Supplemental Fig. S2 .
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