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Cells are like people: depending on their current environment, past experiences and their genetic make-up they will react differently. Treat cells in different ways, and they will produce different cytokines. There are a lot of protocols out there for stimulating cells. Depending on the species of cells you plan to stimulate, different protocols are available and each will result in a unique cytokine secretion profile.
Never fear! For those of you interested in human peripheral blood mononuclear cells (PBMCs), I have broken down the protocols and given you a simple table to follow:
|PROTOCOL SUMMARY||CYTOKINES SECRETED|
|1. Stimulate with immobilised anti-human CD3 antibody, soluble anti-human CD28 antibody, recombinant human IL-2 and IL-4.|
2. Wash and culture with the same IL-2 and IL-4.
3. Harvest and restimulate with PMA, calcium ionophore or ionomycin.
|IL-3, IL-4, IL-5, IL-13 and GM-CSF|
|1. Stimulate with immobilised anti-human CD3 antibody and recombinant human IL-2.|
2. Wash and culture in more IL-2.
3. Harvest and restimulate with PMA, calcium ionophore or ionomycin. Alternatively, restimulate with anti-CD3 and anti-CD28.
|Stimulate with PMA, calcium ionophore or ionomycin.||IL-2, TNF- α, and IFN-γ|
|Stimulate with LPS. A 4-hour stimulation (a) will result in a different profile to a 24-hour stimulation (b).||(a) IL-1α, IL-6, IL-8 and GRO- α
(b) IL-10, MCP-1, MIP-1 α, MCP-3, and MIG
|Prime with recombinant human IFN-γ and LPS.||IL-12|
|Culture for 24 hours (as T cells constitutively produce this cytokine).||RANTES|
More about the players involved
Ionomycin: ionomycin is an ionophore made by Streptomyces conglobatu and is used to raise intracellular levels of calcium (important for NFAT signalling). It also stimulates the production of cytokines like IFN-?, perforin, IL-2 and IL4.
PMA: Phorbol 12-myristate 13-acetate, or PMA to its friends, makes its way into the cell where it activities protein kinase C and is thus a potent tumor inducer. Induces cell proliferation.
IL-2: The growth, proliferation and encourager or differentiation cytokine. IL-4: a potent inducer of T cell differentiation into a Th2 phenotype creating a positive feedback loop.
Anti-CD3 and anti-CD-28: promote CD4 stimulation and expansion.
What protocols do you use? I’ve heard playing Iron Maiden stimulates inflammatory cytokines like nothing else! Or maybe that was ionomycin…