Platelet activation was quantified by the upregulation of their surface markers, CD63 and CD62P, by flow cytometry. Briefly, after separation from whole blood, the platelet suspension was kept at 25°C for 4 hours, followed by re-calcification and incubation with E. coli for 15 minutes at 37°C. Then, platelets were stained for 30 minutes in the dark at 4°C with an antibody mixture containing platelet CD42a-FITC (catalog no. 348083, BD Biosciences, San Jose, CA), CD63-PE-Cy7 (catalog no. 25-0639-42, Invitrogen, Carlsbad, CA), and CD62P-PE (catalog no. 555524, BD Biosciences). After staining, the solution was centrifuged at 250×g, 4°C for 5 minutes. The supernatant was discarded and the pellet was resuspended and fixed in PBS containing 0.1% paraformaldehyde (PFA) and 0.1% bovine serum albumin (PBSA) and stored at 4°C until analysis with flow cytometer Attune NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific) within 24 hours from sampling. Platelets were gated as CD42a+ population (Supplementary Figure 1
Cd42a fitc
Manufactured by BD
Sourced in United States
CD42a-FITC is a fluorescently-labeled monoclonal antibody used for the detection and analysis of the CD42a antigen, which is expressed on the surface of platelets. This product is designed for use in flow cytometry applications.
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Lab products found in correlation
Cd62p pe, by BD (2 mentions)
Cd62 pe, by BD (2 mentions)
Fc500 flow cytometer, by Beckman Coulter (2 mentions)
Cd63 pe cy7, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated pac1 antibody, by BD (1 mentions)
Cd14 pe, by BD (1 mentions)
Dasatinib, by Cayman Chemical (1 mentions)
Nilotinib, by Cayman Chemical (1 mentions)
Biotin conjugated anti rabbit igg, by Merck Group (1 mentions)
Avidin biotin complex kit, by Vector Laboratories (1 mentions)
Anti actin, by Merck Group (1 mentions)
Pac 1 fitc, by BD (1 mentions)
Cd41 pe, by Agilent Technologies (1 mentions)
Sepharose cl 2b, by Merck Group (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
5 protocols using cd42a fitc
1
Platelet Activation Measurement by Flow Cytometry
2
Monocyte-Platelet Aggregation and Activation
3
Platelet and Leukocyte Aggregation Analysis
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The expression of platelet surface P-selectin and leukocyte - platelet heterotypic aggregates were measured by flow cytometry (FC500 flow cytometer, Beckman Coulter). To standardize measurement conditions and to minimize in vitro platelet activation whole blood was fixed for P-selectin measurement and labelled for heterotypic aggregate determination within 2 h after blood collection. After fixation and washing by PBS, samples were labelled by CD42a-FITC and CD62-PE (Becton Dickinson Biosciences) for 20 min, washed twice and analyzed. Whole blood samples were incubated with CD14-PE and CD42a-FITC for 15 min to detect leukocyte-platelet heterotypic aggregates. Results were always compared to samples stained with non-immune IgG that served as isotype controls. Red blood cells were lysed and the samples were washed twice and fixed and subsequently analyzed.
4
Platelet Activation via P-selectin Expression
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Investigation of platelet activation level via surface P-selectin expression on platelets was performed, as previously reported [26 (link)]. Briefly, 40 μL of whole blood samples were fixed in 1 mL 1% PFA and kept at RT for 1 h. Platelets were identified by a FITC-conjugated monoclonal antibody to GPIX (CD42a-FITC, Becton Dickinson, San Jose, CA, USA). Platelet activation was detected by phycoerythrin (PE)-labeled anti-P-selectin (CD62-PE, Becton Dickinson, San Jose, CA, USA). Fixed platelets were incubated with saturating concentrations of antibodies for 20 min in the dark at RT. As a control for immunolabeling with anti-CD62 antibody, platelets were incubated with PE-coupled non-immune mouse IgG1 antibody (Becton Dickinson, San Jose, CA, USA). A total of 10,000 dual-color labeled platelet events were acquired on an FC-500 flow cytometer (Beckman Coulter, Pasadena, CA, USA). Results were expressed as the percentage of double-positive platelets.
5
Platelet activation signaling pathway
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Dasatinib and nilotinib for in vitro experiments were from Cayman Chemical (Ann Arbor, MI, USA). The following directly conjugated monoclonal antibodies were purchased from Becton Dickinson (San Jose, CA, USA): annexin V-FITC, CD41a-PECy5, CD42a-FITC, CD62P-PE, PAC1-FITC. CD41-PE was from DAKO (Glostrup, Denmark). Dimethylsulfoxide (DMSO), Sepharose CL-2B, anti-actin and biotin conjugated anti-rabbit IgG were from Sigma-Aldrich (St. Louis, MO, USA) and convulxin were from Pentapharm (Basel, Switzerland). For western blot we used the following polyclonal antibodies: p-Fyn Y530, p-Lyn Y396, p-Src Y529, and p-Src Y418 (Thermo Fischer Scientific, Rockford, IL, USA); p-Fyn Y416 and p-Lyn Y507 Biorbyt (San Francisco, CA, USA); and Cell Signaling Technology (Leiden, The Netherlands), respectively. Avidin-biotin complex kit was from Vector Laboratories (Burlingame, CA, USA), and ECL reagent was used from Millipore (Billerica, MA, USA).
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