Encyclopedia of Experiments: Immunology
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Transcript
Granulocyte-macrophage colony-stimulating factor, GM-CSF is secreted by GM-CSF-producing T helper or THGM cells. These cells show a low expression of other T helper cell-specific cytokines.
To begin in vitro differentiation of THGM cells, take a T helper cell suspension — isolated from the mouse spleen — containing antigen-activated mature cells and undifferentiated naïve cells.
Add fluorescently-labeled antibodies targeting characteristic surface markers of the naïve and mature cells. Perform fluorescence-activated cell sorting to isolate the naïve T cells.
Plate the cells in a well coated with antibodies that bind to the T cell receptor complex. Add an antibody that binds to the co-stimulatory receptor. The synergistic binding activates the cells.
Add interleukin 7 — a cytokine — inducing differentiation into THGM cells and GM-CSF production. Add phorbol myristate acetate, PMA — a pro-inflammatory compound, ionomycin — a calcium ionophore, and a protein transport inhibitor.
PMA enters the cell and activates a protein kinase. The membrane-permeable ionomycin translocates the calcium ions inside the cell — raising the intracellular calcium concentration.
The protein kinase activation and a high calcium level initiate signals that restimulate the THGM cells — resulting in higher GM-CSF production. The protein transport inhibitor blocks GM-CSF secretion, aiding intracellular detection.
Perform immunofluorescence staining, detecting intracellular cytokines, and analyze the cells using flow cytometry. A majority of cells showing high GM-CSF levels but a low expression of other cytokines indicate successful differentiation into THGM cells.
