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Protocol for assessing and visualizing cell microaggregate formation in whole blood by imaging flow cytometry.

STAR Protoc

January 2025

Heinz-Nixdorf-Chair of Biomedical Electronics, TranslaTUM, School of Computation, Information and Technology, TUM, Germany; Munich Institute of Biomedical Engineering, TUM, Germany. Electronic address:

Blood cell aggregates are clinically useful biomarkers in a number of medical disorders. This protocol provides accurate and quantitative analysis of cell aggregates using a small volume of whole blood and imaging flow cytometry. We describe steps for sample collection, staining, and measurement.

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Platelet cells are essential to maintain haemostasis and play a critical role in thrombosis. They swiftly respond to vascular injury by adhering to damaged vessel surfaces, activating signalling pathways, and aggregating with each other to control bleeding. This dynamic process of platelet activation is intricately coordinated, spanning from membrane receptor maturation to intracellular interactions to whole-cell responses.

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Platelet-Rich Plasma (PRP) is a biological treatment widely used in regenerative medicine for its restorative capacity. Although PRP is typically applied at the time of obtention, long-term storage and preservation could enhance its versatility and clinical applications. The objective of this study was to evaluate the effect of long-term freezing on PRP.

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Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated, fibroinflammatory, multiorgan disease with an obscure pathogenesis. Findings indicating excessive platelet activation have been reported in systemic sclerosis, which is another autoimmune, multisystemic fibrotic disorder. The immune-mediated, inflammatory, and fibrosing intersections of IgG4-RD and systemic sclerosis raised a question about platelets' role in IgG4-RD.

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Transmembrane proteins (TMEMs) are embedded in cell membranes and often have poorly understood functions. Our RNAseq analysis identified 89 tmem genes in zebrafish thrombocytes, leading to further investigation through knockdown experiments and gill bleeding assays. Knockdown of tmem242 significantly increased bleeding, indicating a role in hemostasis.

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