How do you stimulate a Jurkat cell?
Jurkat cels can be activated by exposure to a combination of anti-CD3/anti-CD28 or phytoheamaggutinin (PHA), or the C305 mAb originally developped by Arthur Weiss. IL-2 production should be measured 6 to 12 h later (whatever works best for you). 0.5 to 2 millions cells per ml works.
What is PMA and ionomycin?
PMA activates protein kinase C, while ionomycin is a calcium ionophore, and stimulation with these compounds bypasses the T cell membrane receptor complex and will lead to activation of several intracellular signaling pathways, resulting in T cell activation and production of a variety of cytokines.
What is PMA cell?
PMA, through its activation of PKC, can activate T-cells and stimulate low-level production of IL-2. Phytohemagglutinin can trigger a low level of T-cell activation and IL-2 production by binding non-specifically to the cell surface receptor complex.
What does ionomycin do to cells?
Ionomycin is a membrane permeable calcium ionophore produced by the bacterium Streptomyces conglobatus. This ionophore facilitates the transfer of calcium ions (Ca2+) into and out of cells [1]. Ionomycin can be used to increase intracellular calcium levels triggering cell death through apoptosis and autophagy [1].
Do Jurkat cells express CD28?
CD28-stimulated IL-2 gene expression in Jurkat T cells occurs in part transcriptionally and is cyclosporine-A sensitive. J Immunol. 1992 Apr 15;148(8):2609-16.
How does ionomycin increase calcium?
Ionomycin is a calcium ionophore used to increase intracellular calcium concentration, it does so by: Facilitating the transport of Ca2+ across the plasma membrane. Releasing Ca2+ from its intracellular stores without the involvement of G proteins, resulting in the activation of calcium-activated chlorine channels.
How do you dissolve ionomycin?
1. Add 100 μl of DMSO (not provided) to 1 mg of ionomycin. Mix by vortexing until completely dissolved.
What pathway does PMA activate?
protein kinase C
PMA has traditionally been utilized as a tool to activate the protein kinase C (PKC) signaling pathway.
How does PMA activate PKC?
The third group, atypical isozymes, consists of PKCζ and PKCι[9]. PMA stimulates conventional (α, βI, βII, γ) and novel (δ, ε, η, θ) PKC by mimicking the activating ligand DAG[8]. PKC isoforms of all classifications have been reported in neutrophils from healthy donors[10].
How does ionomycin increase intracellular calcium?
How do you prepare ionomycin?
Preparation of 10 mg/ml (13.38 mM) stock solution Add 100 μl of DMSO (not provided) to 1 mg of ionomycin. Mix by vortexing until completely dissolved. 2. Prepare further dilutions with endotoxin‑free water.
How do you store ionomycin?
Storage/Stability Store the powdered ionomycin product desiccated and protected from light at 2–8 °C. Under these conditions the product is stable for 3 years. Stock solutions in ethanol or DMSO, stored at –20 °C and protected from light, are stable for several months.