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. 2018 Jul 9;8:10300. doi: 10.1038/s41598-018-28211-5

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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MC enables an order of magnitude more parameters than FFC to be analyzed simultaneously during platelet activation. Citrate-anticoagulated blood from the same 3 separate healthy donors in Fig. 2 was simultaneously treated with vehicle or the indicated concentrations of TRAP (A) or ADP (B) for 30 minutes in the presence of a custom platelet-specific, metal-tagged antibody panel. This panel contained antibodies directed against CD41, CD61, CD63, CD9, CD107a, CD154, CD42a, CD42b, CD31, CD36, CD29 and GPVI. Samples were fixed in 1% formaldehyde and analyzed by MC. Data were analyzed using a non-linear fit of log agonist vs. response; variable slope or linear regression in GraphPad Prism 5. Results are expressed as a percentage of the mean metal intensity (MMI) achieved with 200 µM ADP or TRAP (means ± SEM; n = 2/3, with n = 2 accounting for the linear region of the TRAP dose-response [concentrations 1.5–3.5 µM)] and n = 3 accounting for all other concentrations [0–1 µM and 5–200 µM]). Statistical analysis: 1-way ANOVA was used in conjunction with a Dunnett multiple comparison test (all results compared to vehicle control) to indicate statistical significance; *P < 0.05, **P < 0.01 and ***P < 0.001. Abbreviations: ADP, adenosine diphosphate; FFC, fluorescence flow cytometry; MC, mass cytometry; TRAP, thrombin receptor activating peptide.