Encyclopedia of Experiments: Immunology
A subscription to JoVE is required to view this content. Sign in or start your free trial.
Transcript
Megakaryocytes are large, multinucleated cells that give rise to platelets, which are essential for blood coagulation.
To produce platelets in vitro, take a suspension culture of hematopoietic stem and progenitor cells, HSPCs, derived from human umbilical cord blood.
Add thrombopoietin, and incubate in physiological conditions. Thrombopoietin binds to its receptor on the HSPCs, triggering signaling events that lead to the differentiation of the HSPCs into immature megakaryocytes.
The immature cells undergo a maturation process to form matured megakaryocytes. The matured cells are large and contain cytoplasmic secretory granules. They possess a multilobed polyploid nucleus and an invaginated membrane system in the cytoplasm.
Matured megakaryocytes produce long, thin cytoplasmic extensions — proplatelets. The proplatelets — multiple platelet-sized swellings linked by thin cytoplasmic bridges — are released into the medium.
The cytoplasmic bridges break, causing the release of individual platelets. Centrifuge to obtain the platelets as a pellet. Discard the supernatant, and resuspend the pellet in a buffer containing adenosine diphosphate — for platelet activation — and fluorescently labeled antibodies — to identify the activated platelets.
Adenosine diphosphate binds to specific G-protein-coupled receptors on the platelets. The binding initiates a signaling cascade, leading to platelet activation. The activation induces intracellular signaling that activates an integrin complex on the platelet membrane. The fluorescently-labeled antibodies bind to the activated complex.
Analyze the cells using flow cytometry. The presence of an antibody-specific fluorescence signal indicates activated platelets.
