This work was supported by the National Institutes of Health grant (R01 HL086699, HL086699-01A2S1, 1S10RR027327-01) to MM. Our article is partly supported by Carl Zeiss Microimaging LLC.

Reactive oxygen species can be measured in live cells using oxidation sensitive dyes (1 &amp; 2) or using plasmid sensors (3 &amp; 4) by confocal microscopy. 
1. Visualization of cytosolic ROS in J774 cells
<ol>
	<li>Grow J774.1 mouse monocyte-derived macrophages (106 cells/ml) on glass bottom 35-mm dishes (Harvard Apparatus) for 48 h. </li>
	<li>Challenge cells with TLR agonists (2 &mu;g/ml, Lipo-teichoic acid-TLR2 agonist; 10&mu;g/ml, Poly (I:C)-TLR3 agonist; 1 &mu;g/ml LPS-TLR4...

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The method described here allows rapid and quantitative measurement of reactive oxygen species in living cells either using oxidation sensitive dyes or plasmid sensors. Agonists of TLRs (Toll-like receptors) are compounds that stimulate the cells through the TLRs present on the cell surface and trigger the downstream signaling pathways15. In our protocol, we used three different TLR agonists viz., Lipo-teichoic acid-TLR2 agonist; Poly (I:C)-TLR3 agonist; LPS-TLR4 agonist which are reported to induce ROS as a m...

<table border="1">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Attofluor cell chamber</td>
			<td>Invitrogen</td>
			<td>A7816</td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma Aldrich</td>
			<td>A8674</td>
		</tr>
		<tr>
			<td>DMEM low glucose medium</td>
			<td>Invitrogen</td>
			<td>10567-014</td>
		</tr>
		<tr>
			<td>Endothelial growth factor supplement (ECGS)</td>
			<td>Upstate</td>
			<td>02-102</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>12662011</td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Invitrogen</td>
			<td>10131-027</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1393</td>
		</tr>
		<tr>
			<td>H2DCFDA</td>
			<td>Invitrogen</td>
			<td>D-399</td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Sigma Aldrich</td>
			<td>L4516</td>
		</tr>
		<tr>
			<td>LTA</td>
			<td>Sigma Aldrich</td>
			<td>L2515</td>
		</tr>
		<tr>
			<td>MitoSOX Red</td>
			<td>Invitrogen</td>
			<td>M36008</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Invitrogen</td>
			<td>51985091</td>
		</tr>
		<tr>
			<td>Pen/Strep (10x)</td>
			<td>Invitrogen</td>
			<td>15140163</td>
		</tr>
		<tr>
			<td>pHyPer-cyto</td>
			<td>Evrogen</td>
			<td>FP941</td>
		</tr>
		<tr>
			<td>pHyPer-dMito</td>
			<td>Evrogen</td>
			<td>FP942</td>
		</tr>
		<tr>
			<td>Poly(I:C)</td>
			<td>Sigma Aldrich</td>
			<td>P0913</td>
		</tr>
		<tr>
			<td>Prism software 5.0</td>
			<td>GraphPad Software, Inc.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SigmaPlot 11.0</td>
			<td>Systat software, Inc.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (10x)</td>
			<td>Invitrogen</td>
			<td>15400054</td>
		</tr>
		<tr>
			<td>T-25 Flasks</td>
			<td>Corning</td>
			<td>430639</td>
		</tr>
		<tr>
			<td>T-75 Flasks</td>
			<td>BD Falcon</td>
			<td>353136</td>
		</tr>
		<tr>
			<td>96-well TC micro well plate</td>
			<td>BD Falcon</td>
			<td>353072</td>
		</tr>
		<tr>
			<td>Zen 2009 software</td>
			<td>Carl Zeiss 510 Meta confocal microscopy</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

visualization of vascular ca2+ signaling triggered by paracrine derived ros

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Watch this Scientific Journal Video about Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS at JoVE.com

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

visualization-vascular-ca2-signaling-triggered-paracrine-derived

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15.4K vues.  Temple University. Une méthode efficace pour mieux comprendre en visualisant l'induction paracrine dérivés du ROS endothéliales de signalisation Ca2 + est décrite. Cette méthode prend avantage de mesurer paracrine dérivés ROS déclenché la mobilisation de Ca2 + dans les cellules endothéliales vasculaires dans un modèle de co-culture.