ionomycin

Responses are expressed as a fraction of the ionomycin (maximum) response.

Control cells were not incubated with ionomycin.

Ionomycin is an ionophore produced by the bacterium Streptomyces conglobatus.

ATP and ionomycin treatment in the absence of anisomycin only weakly increased p38 phosphorylation.

The PMA and ionomycin control induced apoptosis in both cell types (data not shown).

Upon treatment with the calcium ionophore ionomycin, GFP-NFAT translocates rapidly to the nucleus.

BJAB-B1 cells stimulated with PMA/ionomycin were used as a positive IF control (Figure 7B).

Apoptosis was detected in parental and PLCγ2-deficient cells in response to the control PMA and ionomycin treatment (Fig 5C).

GFP, and PMA/ionomycin activation had little effect on either the frequency or expression levels of GFP positive cells.

The control treatment with PMA and ionomycin induced JNK phosphorylation in both cell types (Fig 3B, lanes 6 and 12).

Following activation with PMA and ionomycin, CMV5-driven expression of GFP increased to 81% of the CD4 +T cells.

Upon activation with PMA/ionomycin, the CD4 +T cells down-regulated CD62L and up-regulated CD25 (Fig.

In in vitro studies, the effect of this necessary calcium signal for activation of genes transcription factor can be induced by the action of calcium ionophores such as ionomycin.

TPA, together with ionomycin, can also be used to stimulate T-cell activation, proliferation, and cytokine production, and is used in protocols for intracellular staining of these cytokines.

GFP, with or without activation with PMA/ionomycin, and expression of GFP was measured by flow cytometry 24 hours later (Fig.

PMA and ionomycin (1 μg/ml) non-specifically reactivates EBV and was used as a positive control for BZLF1 transcript expression in the cell lines.

In some cells, calcium entry is stimulated when the ER stores are artificially emptied by applying the calcium pump inhibitors thapsigargin or 2,5-di- tert- butylhydroquinone, or the calcium ionophore ionomycin.

The cells were then treated with 1 μM ionomycin for 30 minutes, after which time they were either fixed immediately or washed extensively with PBS and incubated for 6 hours in medium lacking the drug ("washout").

Similar results were obtained using intracytoplasmic cytokine staining of the same cells (Figure 3D), in which PMA/ionomycin stimulation strongly induced IFN-γ production from CD69 + /CD8 + lung lymphocytes.

In contrast, both the frequencies and levels of GFP expression were increased in Ad5.CMV5.GFP-transduced Th1 and Th2 lines following activation with PMA/ionomycin, anti-CD3 or OVA peptide.

To test whether PLCγ2 is required for Btk:ER-induced calcium mobilization, parental and PLCγ2-deficient cells expressing Btk:ER were treated with 4-HT and/or anti-μ, or ionomycin as a positive control (Fig 4B).

Consistent with previous data, no calcium mobilization was observed in PLCγ2-deficient cells in response to anti-μ [ 37], but as expected a calcium flux was detected in response to ionomycin, that bypasses receptor-proximal signaling events (Fig 4B).

Statistical Analysis The percent of ionomycin data from the Ca ++elevation experiments and the B/B 0 data from the binding experiments were both subjected to two-way ANOVA analysis, with the independent variables being fluorescent conjugate and dose.

When normal human PBMC were stimulated for 7 days with SEB, the presence of 5A6 anti-CD81 mAb resulted in a modest increase in IL-4-producing T cells, when those cells were restimulated with PMA and ionomycin (Figure 4A).

In addition, using cells expressing wild-type cPLA 2 and cPLA 2 with S505A or S727A mutations, it was found that translocation kinetics and membrane targeting in response to ATP or ionomycin was similar to wild-type cPLA 2 .