Primers used to determine prostaglandin E₂ receptor expression on LAD2 cells by quantitative PCR.

<p>*variant number 5 of the ten existing variants of the receptor.</p><p>Primers used to determine prostaglandin E₂ receptor expression on LAD2 cells by quantitative PCR.</p

EP₂, EP₃, and EP₄ are expressed on human mast cells. At low doses prostaglandin E₂ inhibits mannitol mast cell degranulation.

<p>Real time PCR was performed in LAD2 cells using EP receptor probes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110870#pone-0110870-t001" target="_blank">Table 1</a> (A). The EP receptors expression levels were normalized with the β-actin expression level, EP₁ expression was undetectable. Western blot analysis was carried out with specific antibodies against EP₁, EP₂, EP₃, and EP₄ in whole cell lysates from CD34⁺ derived mast cells and LAD2 cells; blot against β-actin was performed as a loading control (B). PGE₂ titration was carried out before 10% mannitol stimulation in LAD2 cells (C). The experiments are representative of 3 independent assays. Statistical significance (*p≤0.05) is relative to mannitol-stimulated cells.</p

Mast cell degranulation is induced by mannitol in human mast cells.

<p>LAD2 mast cells were stimulated with mannitol (5%, 10%, and 17%) for 30 minutes, and β-hexosaminidase release was measured (A). CD63 staining was performed in LAD2 cells after mannitol stimulation (10%) for 30 minutes. Positive control was conducted with PMA plus ionomycin. Negative control (NC) means isotype staining (B). β-hexosaminidase content was measured in the supernatants from activated LAD2 cells, CD34+ derived human mast cells, and human lung mast cells induced by mannitol (10%) at 37°C for 30 minutes (C). Results are in triplicate and are the mean of three independent experiments expressed as mean ± standard deviation. Statistical significance *p≤0.05, **p≤0.01, ***p≤0.001) is relative to the unstimulated control cells.</p

Calcium mobilization, cytokine production and PGE₂ secretion are induced by mannitol in LAD2 cells.

<p>Determination of calcium flux following mannitol stimulation (10%) was performed in LAD2 cells loaded with Fluo-4 dye, either with calcium containing media (A) or without extracellular calcium (B), as described in material and methods section. After defining basal conditions the stimuli was added (time 0) and measured 10 minutes. Real time polymerase chain reaction was performed in LAD2 cells that were either unstimulated or stimulated with 10% mannitol, using interleukin-8 and tumor necrosis factor alfa as probes. PMA+ Ionomycin (Sigma) were used as positive control stimuli (C). PGE₂ release was measured at different times as indicated in the figure using an ELISA assay. Results are expressed in pg per 1×10⁶ cells (D). Results are triplicates expressed as mean ± standard deviation, and are representative of three independent experiments. Statistical significance (* p≤0.05, **p≤0.01) is relative to unstimulated control cells. RFU: relative fluorescence units.</p