RESUMEN
Suspension cell lines grow free-floating in the cell culture media without any attachment to the culture plate/vessel. Suspension cells typically mimic cells that exist in the circulation of multicellular animals such as mouse and humans. Generally, cell lines derived from the blood such as lymphocytes, megakaryocyte, and neutrophils grow in suspension. These cell lines can be used for experimental studies to understand the biology/biochemistry of cancer cells. In this chapter, procedures for working with suspension cell lines are provided, including protocols for thawing, culturing, and cryopreserving cancer cell lines. Importantly, this chapter demonstrates the best practices required to work with suspension cell lines, to minimize the risk of contaminations from adventitious microorganisms or from other cell lines.
Asunto(s)
Técnicas de Cultivo de Célula , Neoplasias , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Ratones , SuspensionesRESUMEN
Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles that carry bioactive cargo, including proteins, lipids, and nucleic acids. Once taken up by target cells, EVs can modify the physiology of the recipient cells. In past studies, we reported that engagement of the glycophosphatidylinositol-anchored receptor CD24 on B lymphocytes (B cells) causes the release of EVs. However, a potential function for these EVs was not clear. Thus, we investigated whether EVs derived from CD24 or IgM-stimulated donor WEHI-231 murine B cells can transfer functional cargo to recipient cells. We employed a model system where donor cells expressing palmitoylated GFP (WEHI-231-GFP) were cocultured, after stimulation, with recipient cells lacking either IgM (WEHI-303 murine B cells) or CD24 (CD24 knockout mouse bone marrow B cells). Uptake of lipid-associated GFP, IgM, or CD24 by labeled recipient cells was analyzed by flow cytometry. We found that stimulation of either CD24 or IgM on the donor cells caused the transfer of lipids, CD24, and IgM to recipient cells. Importantly, we found that the transferred receptors are functional in recipient cells, thus endowing recipient cells with a second BCR or sensitivity to anti-CD24-induced apoptosis. In the case of the BCR, we found that EVs were conclusively involved in this transfer, whereas in the case in the CD24 the involvement of EVs is suggested. Overall, these data show that extracellular signals received by one cell can change the sensitivity of neighboring cells to the same or different stimuli, which may impact B cell development or activation.