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 agonist) for 6 h at 37&deg;C. 
	Note: Macrophages treated under similar conditions were used for co-culture model Ca2+ mobilization.</li>
	<li>Add the oxidation-sensitive dye H2DCF-DA (Invitrogen) to give a final concentration of 10 &mu;M in ECM (Extracellular medium: 121 mM NaCl, 5 mM NaHCO3, 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 10 mM glucose and 2.0% dextran, pH 7.4; all obtained from Sigma) containing 2%BSA to dishes and incubate cells for 20 min before visualization under confocal microscopy. </li>
	<li>After dye loading, visualize cells placed on a temperature controlled stage of an appropriate imaging system. We use Carl Zeiss LSM 510 Meta confocal imaging system coupled with Argon ion laser source at an excitation of 488 nm for DCF fluorescence using 40x oil objective13 (Fig. 1A and D).</li>
	<li>Analyze images using ImageJ software (1.44), freely available software from the National Institutes of Health (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, <a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a>, 1997-2009).</li>
</ol>
2. Visualization of mROS 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 the cells with TLR ligands to induce mROS production (2 &mu;g/ml, Lipo-teichoic acid-TLR2; 10&mu;g/ml, Poly (I:C)-TLR3; 1 &mu;g/ml LPS-TLR4) for 6 h at 37&deg;C. </li>
	<li>Add mitochondrial superoxide indicator MitoSOX Red (Invitrogen) to give a final concentration of 5 &mu;M in ECM containing 2%BSA to dishes and incubate cells for 30 min at 37&deg;C to allow loading of MitoSOX Red. </li>
	<li>After dye loading, visualize cells on a temperature controlled stage of a Carl Zeiss LSM 510 Meta confocal imaging system couple with Argon ion laser source at an excitation of 561 nm for MitoSOX Red fluorescence using 40x oil objective (Fig 1B and E).</li>
	<li>For control experiments, after dye loading add PBS or DMSO (negative control) or 2&mu;M Antimycin A (as mitochondrial superoxide generator-positive control) to cells prior to image the sample as described in 2.4 (Fig 1C).</li>
	<li>Analyze the images using ImageJ software (1.44).</li>
</ol>
3. Visualization of cytosolic ROS in stable HyPer-cyto MPMVECs
<ol>
	<li>We use pHyPer-cyto (Evrogen) a mammalian expression vector encoding a fluorescent sensor HyPer that localizes in the cytoplasm and specifically senses submicromolar concentration of H2O214.</li>
	<li>Transfect Murine Pulmonary Microvascular Endothelial cells (MPMVECs) with pHyPer-cyto vector using either electroporation or transfection reagents. We use 10 x 106 cells and transfect with 10&mu;g of pHyPer-cyto via electroporation using BioRad Gene pulser electroporator system (250V; 500 &mu;F). Culture MPMVECs in DMEM medium supplemented with 10% FBS, nonessential amino acids, endothelial growth supplement and antibiotics. 
	Note: Following transfection, plate cells at low density to facilitate colony growth.</li>
	<li>Replace medium with complete culture medium supplemented with 500 &mu;g/ml G418, 48 hours after transfection. Screen for clones that have highest percentage of HyPer positive cells and passage HyPer positive clones to increase the number of HyPer positive cells (Fig. 2). 
	Note: We use serial dilution of cells for clonal selection of single cells and screen for stable cells highly expressing HyPer.</li>
	<li>Grow stable HyPer-cyto MPMVECs on 0.2% gelatin coated coverslips (80% confluence). Next day challenge cells with TLR ligands (2 &mu;g/ml, Lipoteichoic acid-TLR2; 10&mu;g/ml, Poly (I:C)-TLR3; 1 &mu;g/ml LPS-TLR4) for 6 h at 37&deg;C. Untreated cells serve as control.</li>
	<li>After treatment, mount the coverslips onto Attofluor cell chamber (Invitrogen). Add 1ml of pre warmed Hanks balanced salt solution (HBSS) to the chamber and place the chamber on a temperature controlled stage of Carl Zeiss LSM 510 Meta confocal imaging system. Visualize and image fluorescence change at an excitation of 488 nm using 40x oil objective (Fig. 3A and D).</li>
	<li>Analyze images using ImageJ software (1.44) (see section 6).</li>
</ol>
4. Visualization of mitochondrial ROS in stable HyPer-dMito MPMVECs
<ol>
	<li>We use pHyPer-dMito (Evrogen) a mammalian expression vector encoding mitochondria-targeted HyPer that localizes in the mitochondria and specifically senses submicromolar concentration of H2O2.</li>
	<li>Transfect MPMVECs with pHyPer-dMito and generate stable HyPer-dMito MPMVECs following steps described in section 3.2-3.3. </li>
	<li>Grow stable HyPer-dMito MPMVECs on 0.2% gelatin coated coverslips (80% confluence).</li>
	<li>Next day challenge cells with TLR ligands (2 &mu;g/ml, Lipoteichoic acid-TLR2; 10&mu;g/ml, Poly (I:C)-TLR3; 1 &mu;g/ml LPS-TLR4) for 6 h at 37&deg;C. Untreated cells serve as negative control. Add 2&mu;M Antimycin A (mitochondrial superoxide inducer-positive control) prior to visualizing sample.</li>
	<li>After treatment, mount the coverslips onto Attofluor cell chamber (Invitrogen). Add 1ml of pre-warmed Hanks balanced salt solution (HBSS) to the chamber and place the chamber on a temperature controlled stage of Carl Zeiss LSM 510 Meta confocal imaging system. Visualize and image fluorescence change at an excitation of 488 nm using 40x oil objective (Fig. 3B, C and E).</li>
	<li>Analyze images using ImageJ software (1.44) (see section 6).</li>
</ol>
5. Co-culture model for visualization of paracrine-derived ROS triggered Ca2+ signaling in endothelial cells
For the visualization and measurement of [Ca2+]i changes we use a Ca2+ sensitive fluorescent dye fluo-4 AM (Invitrogen).
<ol>
	<li>Grow MPMVECs on 0.2% gelatin coated 25mm glass coverslips (80% confluence).</li>
	<li>Incubate the cells grown on coverslips at room temperature for 30 min in ECM containing 2%BSA and 0.003% pluronic acid with 5 &mu;M fluo-4 AM (Invitrogen) final concentration to allow loading of fluo-4 AM. After loading, wash cells with experimental imaging solution (ECM containing 0.25% BSA) containing sulfinpyrazone to minimize dye loss.</li>
	<li>After loading fluo-4, mount coverslips onto Attofluor cell chamber (Invitrogen). Add 800&mu;l of pre warmed experimental imaging solution to the chamber and place it on a temperature controlled stage of Carl Zeiss LSM 510 Meta confocal imaging system.</li>
	<li>In a separate tube label the nonactivated or activated macrophages isolated from either wild-type or gp91phox-/- mice (treated with LPS for 6 hr) with cell tracker red CMTPX (500ng/ml) (Invitrogen) in ECM containing 2%BSA and 0.003% pluronic acid. Wash and resuspend cells with 200&mu;l of experimental imaging solution. </li>
	<li>Add labeled macrophages on to fluo-4 AM loaded MPMVECs in the chamber assembly.</li>
	<li>Record the green and red fluorescence every 3s for 10-20 min using Carl Zeiss LSM510 Meta confocal imaging system with a Argon ion laser source with excitation at 488 and 561 nm for fluo-4 AM and cell tracker red fluorescence respectively ( Fig.4 A-C). 
	Note: Alternatively, J774.1 cell line could be used to elicit MPMVECs Ca2+ mobilization.</li>
	<li>Analyze fluo-4 fluorescence intensity changes using ImageJ software (1.44) (see section 6).</li>
</ol>
6. Data analysis using ImageJ software
Transfer the images obtained from the confocal system as a .lsm format file for data analysis. ZEN 2009 and ZEN 2010 software provided with LSM 510 Meta confocal microscope or LSM 710 multiphoton confocal microscope respectively saves files in .lsm format. We recommend using this native .lsm files for image analysis as original quality is retained. This file format can be directly opened in image analysis program Image J. The following steps provide an example quantitative analysis of single image. No additional plugins are required for this data analysis.
<ol>
	<li>Open .lsm or .TIFF file in Image J program using FILE&gt;OPEN</li>
	<li>Click the tab IMAGE&gt;COLOR&gt;SPLIT CHANNELS and split the green and DIC channels. Use the image from the green channel for subsequent analysis.</li>
	<li>Double click and select the 'Elliptical brush selection" tab and set the pixel value to 20.</li>
	<li>For Hyper-cyto images, click on a single cell to define the circular area of measurement. Then press "M" key to measure the intensity of the selected area. The values are retained in a new results window.</li>
	<li>Next by holding "Shift" key click on a different cell to select another area, followed by pressing "M" key. The intensity is recorded in the result window. Repeat the steps 4-5 on 50 different cells.</li>
	<li>For Hyper d-mito images, set the pixel value to 10 in the Elliptical brush selection. Region of interest (ROI) has to be manually traced including the mitochondria of particular cells.</li>
	<li>Using freehand, trace the region of around the mitochondria and then press "M". Repeat the steps 6-7 on 50 different cells.</li>
	<li>Copy the mean values from the results window and paste in a Excel sheet or any software to plot the results. We use either SigmaPlot or Graphpad PRISM to plot the data as graph.</li>
</ol>
7. Representative Results
Figure 1 shows cytosolic and mitochondrial ROS production upon challenge with TLR ligands. Representative confocal images show increase of DCF (cytosolic) and MitoSOX Red (mitochondria) fluorescence after 6 hr of stimulation. Fluorescence changes were normalized and expressed as fold change.
Figure 2 shows the generation of stable HyPer-cyto and HyPer-dMito endothelial cells. Mouse endothelial cells were transfected with pHyPer-cyto or pHyPer-dMito plasmid constructs via electroporation. Cells stably expressing plasmids were selected with G418 for two weeks. After selection, diluted cells were seeded in 96 well plates. Individual colonies were isolated and imaged for homogeneous expression.
Figure 3 shows the measurement of endothelial cytosolic and mitochondrial H2O2 levels. Representative confocal images show increase of HyPer-cyto (cytosolic) and HyPer-dMito (mitochondria) fluorescence after 6 hr of stimulation. Fluorescence intensity changes were normalized and expressed as fold change.
Figure 4 shows the assessment of macrophage-derived ROS induced endothelial cytosolic Ca2+ mobilization. LPS-stimulated macrophages were added onto pulmonary microvascular endothelial cells (PMVEC) that had been previously loaded with the intracellular Ca2+ ([Ca2+]i) indicator dye Fluo-4. Application of wild type LPS-activated macrophages evoked an [Ca2+]i rise in PMVECs that was attenuated by knockout of functional NADPH oxidase (gp91phox-/-).
<img src="/files/ftp_upload/3511/3511fig1.jpg" alt="Figure 1" /> 
Figure 1. Visualization of macrophage generated cellular ROS and mitochondrial superoxide. J774.1 cells were challenged with TLR2, 3 and 4 ligands. (2 &mu;g/ml, Lipoteichoic acid-TLR2; 10&mu;g/ml, Poly (I:C)-TLR3; 1 &mu;g/ml LPS-TLR4) for 6 h at 37&deg;C. After treatment cells were loaded with (A) cellular ROS indicator (H2DCF-DA; green fluorescence) or (B) mitochondrial superoxide indicator (MitoSOX Red; red fluorescence). (C) Antimycin A was used as a positive control for mitochondrial ROS production. Fluorescence intensity changes were assessed using confocal microscopy. (D) and (E) Quantitation of mean fluorescence change.
<img src="/files/ftp_upload/3511/3511fig2.jpg" alt="Figure 2" /> 
Figure 2. Schematic representation of generating stable expression of endothelial cell cytosolic and mitochondrial ROS indicators. 
<img src="/files/ftp_upload/3511/3511fig3.jpg" alt="Figure 3" /> 
Figure 3. Visualization of endothelial cytosolic and mitochondrial H2O2 levels. MPMVECs stably expressing (A) HyPer-cyto or (B) HyPer-dMito were challenged with TLR2, 3 and 4 ligands (2 &mu;g/ml, Lipoteichoic acid-TLR2; 10&mu;g/ml, Poly (I:C)-TLR3; 1 &mu;g/ml LPS-TLR4) for 6 h at 37&deg;C. (C) Antimycin A was used as a positive control for mitochondrial ROS production. After treatment, fluorescence intensity changes were assessed using confocal microscopy. (D) and (E) Quantitation of mean fluorescence change.
<img src="/files/ftp_upload/3511/3511fig4.jpg" alt="Figure 4" /> 
Figure 4. Macrophage-derived ROS elicit impaired endothelial cell Ca2+ mobilization. (A) Schematic representation of endothelial cells/macrophage co-culture model study. (B) Freshly isolated murine macrophages from both WT and gp91phox null mice (The Jackson Laboratory) were activated by 1 &mu;g/ml LPS for 6 h. Macrophages were labeled with cell-tracker Red (red) and added onto Fluo-4 loaded PMVECs (green) to assess paracrine O2&bull;- signaling. (C) LPS-activated macrophages isolated from WT mice triggered large and rapid Ca2+ mobilization in PMVECs compared to macrophages from gp91phox null mice. Validation of macrophage activation has been demonstrated previously.

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	<li>Madesh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+role+for+superoxide+in+InsP3+receptor-mediated+mitochondrial+dysfunction+and+endothelial+apoptosis.">Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis.</a> J. Cell. Biol. 170, 1079-1090 (2005).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiol. Rev. 82, 47-95 (2002).</li><li>Hamanaka, R. B., Chandel, N. 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No conflicts of interest declared.

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 ...

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15.4K Views.  Temple University. 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.

Video: Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that has emerged as an essential model for regeneration studies. Little is known about how revascularization occurs in the context of regeneration in these animals. Here we report a simple method for visualization of the vasculature in axolotl via perfusion of 1,1&#8217;-Dioctadecy-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI). DiI is a lipophilic carbocyanine dye that inserts into the plasma membrane of endothelial cells instantaneously. Perfusion is done using a peristaltic pump such that DiI enters the circulation through the aorta. During perfusion, dye flows through the axolotl&#8217;s blood vessels and incorporates into the lipid bilayer of vascular endothelial cells upon contact. The perfusion procedure takes approximately one hour for an eight-inch axolotl. Immediately after perfusion with DiI, the axolotl can be visualized with a confocal fluorescent microscope. The DiI emits light in the red-orange range when excited with a green fluorescent filter. This DiI perfusion procedure can be used to visualize the vascular structure of axolotls or to demonstrate patterns of revascularization in regenerating tissues.

DiI Perfusion as a Method for Vascular Visualization in Ambystoma mexicanum

Using a lipophilic 1,1&#39;-Dioctadecy-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI) staining technique, Ambystoma mexicanum can undergo vascular perfusion to allow for easy visualization of the vasculature.

Perfusion techniques have been used for centuries to visualize the circulation of tissues. Axolotl (Ambystoma mexicanum) is a species of salamander that ...

Visualization of DNA Compaction in Cyanobacteria by High-voltage Cryo-electron Tomography

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.

This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

A Proximal Culture Method to Study Paracrine Signaling Between Cells

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Paracrine or juxtacrine signaling mediates such interactions. The use of a conditioned medium and coculture studies are the most common methods to discriminate between these two types of interactions. However, the effect of localized high concentrations of secreted factors in the microenvironment during the paracrine interactions is not accurately recapitulated by conditioned medium and, thus, may lead to imprecise conclusions. To overcome this problem, we have devised a proximal culture method to study paracrine signaling. The two cell types are grown on either surface of a 10 &#181;m-thick polycarbonate membrane with 0.4 &#181;m pores. The pores allow the exchange of secreted factors and, at the same time, inhibit juxtacrine signaling. The cells can be collected and lysed at the endpoint to determine the effects of the paracrine signaling. In addition to allowing for localized concentration gradients of secreted factors, this method is amenable to experiments involving prolonged periods of culture, as well as the use of inhibitors. While we use this method to study the interactions between ovarian cancer cells and the mesothelial cells they encounter at the site of metastasis, it can be adapted to any two adherent cell types for researchers to study paracrine signaling in various fields, including tumor microenvironment, immunology, and development.

Paracrine and juxtacrine cellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and development. Here, a proximal culture method is used to study paracrine signaling where the localized concentrations of the secreted factors are maintained while preventing direct cellular contact.

Intercellular interactions play an important role in many biological processes, including tumor progression, immune responses, angiogenesis, and ...

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires careful and in-depth analyses and quantification to capture as much information about the underlying events as possible. Spatial, temporal and intensity parameters intrinsic to Ca2+ signals such as frequency, duration, propagation, velocity and amplitude may provide some biological information required for intracellular signalling. High-resolution Ca2+ imaging typically results in the acquisition of large data files that are time consuming to process in terms of translating the imaging information into quantifiable data, and this process can be susceptible to human error and bias. Analysis of Ca2+ signals from cells in situ typically relies on simple intensity measurements from arbitrarily selected regions of interest (ROI) within a field of view (FOV). This approach ignores much of the important signaling information contained in the FOV. Thus, in order to maximize recovery of information from such high-resolution recordings obtained with Ca2+dyes or optogenetic Ca2+ imaging, appropriate spatial and temporal analysis of the Ca2+ signals is required. The protocols outlined in this paper will describe how a high volume of data can be obtained from Ca2+ imaging recordings to facilitate more complete analysis and quantification of Ca2+ signals recorded from cells using a combination of spatiotemporal map (STM)-based analysis and particle-based analysis. The protocols also describe how different patterns of Ca2+ signaling observed in different cell populations in situ can be analyzed appropriately. For illustration, the method will examine Ca2+ signaling in a specialized population of cells in the small intestine, interstitial cells of Cajal (ICC), using GECIs.

Applications of Spatio-temporal Mapping and Particle Analysis Techniques to Quantify Intracellular Ca2+ Signaling In Situ

Genetically encoded Ca2+ indicators (GECIs) have radically changed how in situ Ca2+ imaging is performed. To maximize data recovery from such recordings, appropriate analysis of Ca2+ signals is required. The protocols in this paper facilitate the quantification of Ca2+ signals recorded in situ using spatiotemporal mapping and particle-based analysis.

Ca2+ imaging of isolated cells or specific types of cells within intact tissues often reveals complex patterns of Ca2+ signaling. This activity requires ...