The research is supported by National Institutes of Health grants AI-52381, CA138623 and Kentucky Lung Cancer Research Board and institutional support from James Graham Brown Cancer Center.

Methodology
Description of Microscope
Live cell imaging experiments performed using TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300. The microscope equipped with heating stage. A cool snap HQ digital B/W CCD (Roper Scientific) camera and LAMDA 10-2 optical filter changer (Sutter instrument company) is attached to microscope. Excitation and emission wavelengths are controlled with filter wheels and controlled by Lamba 10-2 filter whee...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifaceted+roles+of+beta-arrestins+in+the+regulation+of+seven-membrane-spanning+receptor+trafficking+and+signalling.">Multifaceted roles of beta-arrestins in the regulation of seven-membrane-spanning receptor trafficking and signalling.</a> Biochem J. 375, 503-515 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-arrest	or.">Beta-arrest	or.</a> Nature. 383, 447-450 (1996).</li><li>Serhan, C. N., Haeggstrom, J. Z., Leslie, C. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLT1+and+BLT2:+the+leukotriene+B(4)+receptors.">BLT1 and BLT2: the leukotriene B(4) receptors.</a> Prostaglandins Leukot Essent Fatty Acids. 69, 123-134 (2003).</li><li>Toda, A., Yokomizo, T., Shimizu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukotriene+B4+receptors.">Leukotriene B4 receptors.</a> Prostaglandins Other Lipid Mediat. 68-69, 575-585 (2002).</li><li>Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+leukotriene+B(4)+receptor+in+mice+reveals+its+role+in+inflammation+and+platelet-activating+factor-induced+anaphylaxis.">Targeted disruption of the leukotriene B(4) receptor in mice reveals its role in inflammation and platelet-activating factor-induced anaphylaxis.</a> J Exp Med. 192, 433-438 (2000).</li><li>Subbarao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+leukotriene+B4+receptors+in+the+development+of+atherosclerosis:+potential+mechanisms.">Role of leukotriene B4 receptors in the development of atherosclerosis: potential mechanisms.</a> Arterioscler Thromb Vasc Biol. 24, 369-375 (2004).</li><li>Jala, V. R., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukotrienes+and+atherosclerosis:+new+roles+for+old+mediators.">Leukotrienes and atherosclerosis: new roles for old mediators.</a> Trends Immunol. 25, 315-322 (2004).</li><li>Heller, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+atherogenesis+in+BLT1-deficient+mice+reveals+a+role+for+LTB4+and+BLT1+in+smooth+muscle+cell+recruitment.">Inhibition of atherogenesis in BLT1-deficient mice reveals a role for LTB4 and BLT1 in smooth muscle cell recruitment.</a> Circulation. 112, 578-586 (2005).</li><li>Miyahara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+leukotriene+B4+receptor+1+in+allergen-induced+airway+hyperresponsiveness.">Requirement for leukotriene B4 receptor 1 in allergen-induced airway hyperresponsiveness.</a> Am J Respir Crit Care Med. 172, 161-167 (2005).</li><li>Terawaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absence+of+leukotriene+B4+receptor+1+confers+resistance+to+airway+hyperresponsiveness+and+Th2-type+immune+responses.">Absence of leukotriene B4 receptor 1 confers resistance to airway hyperresponsiveness and Th2-type immune responses.</a> J Immunol. 175, 4217-4225 (2005).</li><li>Shao, W. H., Del Prete, A., Bock, C. B., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+leukotriene+B4+receptors+BLT1+and+BLT2:+a+critical+role+for+BLT1+in+collagen-induced+arthritis+in+mice.">Targeted disruption of leukotriene B4 receptors BLT1 and BLT2: a critical role for BLT1 in collagen-induced arthritis in mice.</a> J Immunol. 176, 6254-6261 (2006).</li><li>Kim, N. D., Chou, R. C., Seung, E., Tager, A. M., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+requirement+for+the+leukotriene+B4+receptor+BLT1+for+neutrophil+recruitment+in+inflammatory+arthritis.">A unique requirement for the leukotriene B4 receptor BLT1 for neutrophil recruitment in inflammatory arthritis.</a> J Exp Med. 203, 829-835 (2006).</li><li>Jala, V. R., Shao, W. H., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation-independent+beta-arrestin+translocation+and+internalization+of+leukotriene+B4+receptors.">Phosphorylation-independent beta-arrestin translocation and internalization of leukotriene B4 receptors.</a> J Biol Chem. 280, 4880-4887 (2005).</li><li>Jala, V. R., Haribabu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+analysis+of+G+protein-coupled+receptor+signaling+in+live+cells.">Real-time analysis of G protein-coupled receptor signaling in live cells.</a> Methods Mol Biol. 332, 159-165 (2006).</li><li>Del Prete, A., A, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+dendritic+cell+migration+and+adaptive+immune+response+by+leukotriene+B4+receptors:+a+role+for+LTB4+in+up-regulation+of+CCR7+expression+and+function.">Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function.</a> Blood. 109, 626-631 (2007).</li><li>Salogni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin+A+induces+dendritic+cell+migration+through+the+polarized+release+of+CXC+chemokine+ligands+12+and+14.">Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14.</a> Blood. 113, 5848-5856 (2009).</li><li>Boudreau, J., Koshy, S., Cummings, D., Wan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+myeloid+dendritic+cells+from+bone+marrow+precursors.">Culture of myeloid dendritic cells from bone marrow precursors.</a> J Vis Exp. , (2008).</li></ol>

No conflicts of interest declared.

Live cell imaging is a powerful tool to demonstrate the function and interactions of specific proteins as they occur in real-time. The methods described in this manuscript clearly show that LTB4 can induce rapid migration of dendritic cells. These methods not only expand the aspects of LTB4 function to diverse cell types, they allow similar methods to be applied to a variety of other chemokines and testing their efficacy as chemotactic agents on different leukocyte sub populations. Fluorescence ima...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cell lines:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rat Basophilic Leukomia Cell line (RBL-2H3) or HEK293 cells.</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>CRL-2256</td>
<td>&nbsp;</td>
<tr>
<td>Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Delbecco's modified Eagle&#x2019;s Medium (DMEM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11995</td>
<td>&nbsp;</td>
<tr>
<td>Phenol red free RPMI or DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11835-030</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16000-044</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine (200 mM)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin (10000 U/mL)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>HEPES (1M)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15630</td>
<td>&nbsp;</td>
<tr>
<td>Others:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>35 mm sterile glass coverslip-bottomed Fluoro dishes (0.17 mm thick) (WillCo-dish)</td>
<td>&nbsp;</td>
<td>WPI</td>
<td>FD35-100</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Gene Pulser Cuvette (0.4 cm electrode gap) (Bio-Rad)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>16552088</td>
<td>&nbsp;</td>
<tr>
<td>Instruments/software:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gene Pulser II electroporater </td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>TE-FM Epi-Fluorescence system attached to Nikon Inverted Microscope Eclipse TE300</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph Software</td>
<td>&nbsp;</td>
<td>Universal Imaging</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vertical Micro-pipette puller</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Forge M-900</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hadraulic Micromanipulator MO-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Coarse Manual Manipulator, MN-188NE</td>
<td>&nbsp;</td>
<td>Narishige International </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA constructs:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>cDNA of G-Protein coupled receptor tagged with red fluorescence protein at C-terminus (hBLT1-RFP)</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td> cDNA of cytosolic protein tagged with GFP (&beta;-arrestin1-GFP in present study).</td>
<td>&nbsp;</td>
<td>Jala et al 2005</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

real-time imaging of leukotriene b4 mediated cell migration and blt1 interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of &beta;-arrestins3. &beta;-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization 4-6.
Leukotriene B4 (LTB4) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB4 receptor 1 (BLT1; a high affinity receptor) and LTB4 receptor 2 (BLT2; a low affinity receptor)7-9. The LTB4-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis10-17. The current paper describes the methodologies developed to monitor LTB4-induced leukocyte migration and the interactions of BLT1 with &beta;-arrestin and , receptor translocation in live cells using microscopy imaging techniques18-19.
Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described 20-21. These cells were tested in live cell imaging methods to demonstrate LTB4 induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and &beta;-arrestin1 tagged with green fluorescent protein (&beta;-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.

Watch this Scientific Journal Video about Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with Î²-arrestin at JoVE.com

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &amp;#946;-arrestin

This paper describes the methodology to determine the chemotactic response of leukocytes to specific ligands and identify interactions between the cell surface receptors and cytosolic proteins using live cell imaging techniques.

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with &beta;-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to ...

real-time-imaging-leukotriene-b4-mediated-cell-migration-blt1

Research

JoVE Journal

Immunology and Infection

12.7K vues.  University of Louisville. Ce document décrit la méthodologie pour déterminer la réponse chimiotactique des leucocytes à des ligands spécifiques et d'identifier les interactions entre les récepteurs de surface cellulaire et des protéines cytosoliques en utilisant des techniques d'imagerie cellulaire direct.