Flow cytometry reagents such as anti-CD61/PErCP, anti-CD61/PE, anti-CD61/FITC, anti-CD62/PE, PAC-1/FITC, and Cellfix were obtained from Becton Dickinson (San Diego, CA, USA). ADP and collagen were purchased in Chrono-Log (Havertown, PA, USA). All other chemicals were of analytical grade or highest quality available products. Human sP-selectin ELISA kit (Invitrogen).
Anti cd61 pe
Manufactured by BD
Sourced in United States
Anti-CD61-PE is a fluorescently-labeled antibody that binds to the CD61 antigen expressed on the surface of platelets. It is designed for use in flow cytometry applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd61 fitc, by BD (4 mentions)
Cellfix, by BD (2 mentions)
Anti cd62p pe, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Anticd62 pe, by BD (1 mentions)
Human sp selectin elisa kit, by Thermo Fisher Scientific (1 mentions)
Anti cd61 percp, by BD (1 mentions)
Pac 1 fitc, by BD (1 mentions)
Anti cd45 pe, by BD (1 mentions)
Isotype control, by BD (1 mentions)
Silybin, by Merck Group (1 mentions)
Silychristin, by Merck Group (1 mentions)
Igg1 pe, by BD (1 mentions)
Lsrfortessa cell analyzer, by BD (1 mentions)
Huvecs, by Cambrex (1 mentions)
Rivaroxaban, by Bayer (1 mentions)
Thrombin, by Prolytix (1 mentions)
Plasmin, by Prolytix (1 mentions)
Fviia, by Prolytix (1 mentions)
Trypsin, by Prolytix (1 mentions)
8 protocols using anti cd61 pe
1
Flow Cytometry Reagents for Platelet Analysis
2
Flavonolignans Modulate Platelet Function
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Interleukin-1 beta was purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Dimethyl sulfoxide (DMSO), Tris and the flavonolignans (silybin, silychristin and silydianin (Figure S1 ) were all obtained from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Flow cytometry reagents: anti-CD61/FITC, anti-CD61/PE, anti-CD45/PE, isotype controls, BD FACSTM Lysing Solution and CellFix were all obtained from Becton Dickinson (San Diego, CA, USA). All of the other chemicals were of reagent grade or the highest quality available.
3
Flow Cytometric Immunophenotyping of Platelets
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SET-2 or MEG-01 cells were harvested, washed with Dulbecco’s PBS, resuspended in 100 μL of Dulbecco’s PBS and stained with anti-CD41a V450 (1:20; BD Biosciences 561425) and anti-CD61 PE (1:20; BD Biosciences 555754) for 30 min in the dark at RT. After incubation, the cells were washed with Dulbecco’s PBS, resuspended in 400 to 500 μL of FACS buffer (0.01% sodium azide, 0.1% BSA in Dulbecco’s PBS) and analysed on a BD LSRFortessa™ cell analyzer (BD Biosciences). Data were analysed using FlowJo Version 10. V450 IgG1 (1:20; BD Biosciences 560373) and PE IgG1 (1:20; BD Biosciences 555749) were used as isotype controls. Platelet-like particles were gated using FSC and SSC parameters determined from platelets isolated from human peripheral blood [13 (link)].
4
Endothelial Cell Assays with Coagulation Factors
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HUVECs were purchased from Cambrex Bio Science (Charles City, IA, USA) and maintained as previously described [48 (link),49 (link)]. Abnova (Taipei, Taiwan) and Bayer HealthCare (Leverkusen, Germany) provided tumor necrosis factor (TNF) and rivaroxaban, respectively. Collagen and CSL were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Calbiochem-Novabiochem Corp. provided U46619 (San Diego, CA, USA). The following substances were purchased from Haematologic Technologies: plasmin, FVIIa, FX, FXa, activated protein C, tPA, trypsin, and thrombin (Essex Junction, VT, USA). Fisher Diagnostics provided reagents for thromboplastin aPTT and PT assays (Middletown, VA, USA). The chromogenic substrates were all purchased from Chromogenix AB (Mölndal, Sweden) and were designated S-2222 for trypsin, S-2228 for tPA, S2238 for thrombin, S-2251 for plasmin, S-2366 for activated protein C, and S-2765 for FXa. BD Pharmingen (BD Biosciences, San Diego, CA, USA) provided the anti-CD61-FITC, anti-CD62PPE, anti-PAC-1, and anti-CD61-PE antibodies. Santa Cruz Biotechnology provided anti-tissue factor (TF) antibodies (Santa Cruz, CA, USA).
5
CD61 Expression Analysis in MEG-01 and CMY Cells
6
Platelet Activation Assay Protocol
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Adenosine 5′- diphosphate (ADP), collagen and prostaglandin E1 (PGE-1) were obtained from Sigma-Aldrich (St. Louis, Missouri/MO, U.S.A). Convulxin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Sodium chloride (p.a.) was obtained from Arquimed (Santiago, Chile). Antiphospho (S660)-PKC-β2 and antiphospho (Tyr753)-PLC-γ2 antibodies were obtained from Santa Cruz (Biotechnology, CA, USA). Anti γ-tubulin monoclonal antibody (4D11) was obtained from Thermo Scientific (Thermo Scientific, Pierce, Rockford, IL, USA). Antibodies (anti-CD62P-PE, anti-CD61-FITC, anti-GPIIb/IIIa-FITC PAC-1 and anti-CD61-PE) were obtained from BD Pharmingen (BD Biosciences, San Diego, CA, USA).
7
Platelet-Derived EVs Modulate Neutrophil Activation
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Whole blood from the same four healthy donors as above was stimulated for 15 min with 50 μL of EVs isolated from platelets from the same donor stimulated with HEPES, thrombin, or M1 protein, as described above. M1 protein (2.5 μg/mL) and thrombin (1U/mL) in combination with the anticoagulant peptide Gly-Pro-Arg-Pro (1.25 mg/mL) were used as positive controls for platelet activation. HEPES buffer alone was used to determine the background platelet and leukocyte activation. After stimulation, the samples were incubated for 15 min protected from light with anti-CD61-PE (1:10) (BD Biosciences, clone VI-PL2) to detect platelets associated with neutrophils and anti-CD11b-PerCP (1:10) (BD Biosciences, clone ICRF44) to determine neutrophil activation. The samples were acquired on an Accuri C6 Plus Flow Cytometer (BD Biosciences), and the data were analyzed using C6 Plus Software. The neutrophils were gated based on size and granularity, and the CD61 and CD11b intensity within the neutrophil gate was analyzed in histograms.
8
CD61 Expression Analysis in MEG-01 and CMY Cells
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MEG-01 and CMY cell lines were transfected with siRNA and incubated for 4 and 5 days, respectively. Once cells were harvested and washed with 1X PBS, cells were incubated with anti-CD61-PE (Clone: VI-PL2, BD Biosciences, San Jose, CA) for 30 min at RT, washed with 1X PBS, and resuspended in 1X PBS for analyzation. The analysis was performed on BD Bioscience FACS Canto II Flow Cytometer.
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