Name: a flow cytometry-based assay to identify compounds that disrupt binding of fluorescently-labeled cxc chemokine ligand 12 to cxc chemokine receptor 4
Uploaded: 2018-03-10T13:00:00
Description: Watch this Scientific Journal Video about A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4 at JoVE.c

The authors would like to thank Eric Fonteyn for excellent technical assistance. This work has been supported by the KU Leuven (grant no. PF/10/018), Fonds voor Wetenschappelijk Onderzoek (FWO, grant no. G.485.08) and the Fondation Dormeur Vaduz.

1. Maintenance of Cell Culture
NOTE: All steps described under 1 and 2 are carried out under sterile conditions in a laminar flow cabinet.
<ol>
	<li>Grow cells in T75 culture flasks at 37 &#176;C and 5% CO2 in a humidified incubator. 
	NOTE: In this assay, Jurkat cells (i.e., human leukemic T lymphocyte cells that endogenously express CXCR417) are used. Expression of CXCR4 at the cell surface should be evaluated throughout cell culturing by me...

<ol>
	<li>Fredriksson, R., Lagerstrom, M. C., Lundin, L. G., Schioth, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+G-protein-coupled+receptors+in+the+human+genome+form+five+main+families.+Phylogenetic+analysis,+paralogon+groups,+and+fingerprints.">The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints.</a> Mol Pharmacol. 63, 1256-1272 (2003).</li><li>Milligan, G., Kostenis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotrimeric+G-proteins:+A+short+history.">Heterotrimeric G-proteins: A short history.</a> Br J Pharmacol. 147 Suppl 1, S46-S55 (2006).</li><li>Oldham, W. M., Hamm, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotrimeric+G+protein+activation+by+G-protein-coupled+receptors.">Heterotrimeric G protein activation by G-protein-coupled receptors.</a> Nat Rev Mol Cell Biol. 9, 60-71 (2008).</li><li>Tuteja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+G+protein+coupled+receptors.">Signaling through G protein coupled receptors.</a> Plant Signal Behav. 4, 942-947 (2009).</li><li>Neves, S. R., Ram, P. T., Iyengar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein+pathways.">G protein pathways.</a> Science. 296, 1636-1639 (2002).</li><li>Gurevich, E. V., Tesmer, J. J., Mushegian, A., Gurevich, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G+protein-coupled+receptor+kinases:+more+than+just+kinases+and+not+only+for+GPCRs.">G protein-coupled receptor kinases: more than just kinases and not only for GPCRs.</a> Pharmacol Ther. 133, 40-69 (2012).</li><li>Smith, J. S., Rajagopal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-arrestins:+Multifunctional+regulators+of+G+protein-coupled+receptors.">The beta-arrestins: Multifunctional regulators of G protein-coupled receptors.</a> J Biol Chem. 291, 8969-8977 (2016).</li><li>Pierce, K. L., Premont, R. T., Lefkowitz, R. 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C., Farzan, M., Choe, H., Parolin, C., Clark-Lewis, I., Sodroski, J., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lymphocyte+chemoattractant+SDF-1+is+a+ligand+for+LESTR/fusin+and+blocks+HIV-1+entry.">The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry.</a> Nature. 382, 829-833 (1996).</li><li>Flomenberg, N., DiPersio, J., Calandra, G. <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+CXCR4+chemokine+receptor+blockade+using+AMD3100+for+mobilization+of+autologous+hematopoietic+progenitor+cells.">Role of CXCR4 chemokine receptor blockade using AMD3100 for mobilization of autologous hematopoietic progenitor cells.</a> Acta Haematol. 114, 198-205 (2005).</li><li>Domanska, U. M., Kruizinga, R. C., Nagengast, W. B., Timmer-Bosscha, H., Huls, G., de Vries, E. G., Walenkamp, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+CXCR4/CXCL12+axis+in+oncology:+No+place+to+hide.">A review on CXCR4/CXCL12 axis in oncology: No place to hide.</a> Eur J Cancer. 49, 219-230 (2013).</li><li>Tsou, L. K., Huang, Y. H., Song, J. S., Ke, Y. Y., Huang, J. K., Shia, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+CXCR4+antagonists+in+stem+cell+mobilization,+HIV+infection,+ischemic+diseases,+and+oncology.">Harnessing CXCR4 antagonists in stem cell mobilization, HIV infection, ischemic diseases, and oncology.</a> Med Res Rev. , (2017).</li><li>Schols, D., Struyf, S., Van Damme, J., Este, J. A., Henson, G., De Clercq, E. <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+T-tropic+HIV+strains+by+selective+antagonization+of+the+chemokine+receptor+CXCR4.">Inhibition of T-tropic HIV strains by selective antagonization of the chemokine receptor CXCR4.</a> J Exp Med. 186, 1383-1388 (1997).</li><li>Van Hout, A., D'Huys, T., Oeyen, M., Schols, D., Van Loy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cell-based+assays+for+the+identification+and+evaluation+of+competitive+CXCR4+inhibitors.">Comparison of cell-based assays for the identification and evaluation of competitive CXCR4 inhibitors.</a> PLoS One. 12, e0176057 (2017).</li><li>De Clercq, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AMD3100+story:+the+path+to+the+discovery+of+a+stem+cell+mobilizer+(Mozobil).">The AMD3100 story: the path to the discovery of a stem cell mobilizer (Mozobil).</a> Biochem Pharmacol. 77, 1655-1664 (2009).</li><li>Debnath, B., Xu, S., Grande, F., Garofalo, A., Neamati, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+inhibitors+of+CXCR4.">Small molecule inhibitors of CXCR4.</a> Theranostics. 3, 47-75 (2013).</li><li>Woodard, L. E., Nimmagadda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CXCR4-based+imaging+agents.">CXCR4-based imaging agents.</a> J Nucl Med. 52, 1665-1669 (2011).</li><li>Wang, Y., Xie, Y., Oupicky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+CXCR4/CXCL12+Chemokine+Axis+in+Cancer+Drug+Delivery.">Potential of CXCR4/CXCL12 Chemokine Axis in Cancer Drug Delivery.</a> Curr Pharmacol Rep. 2, 1-10 (2016).</li><li>Nimmagadda, S., Pullambhatla, M., Stone, K., Green, G., Bhujwalla, Z. M., Pomper, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+CXCR4+receptor+expression+in+human+cancer+xenografts+with+[64Cu]AMD3100+positron+emission+tomography.">Molecular imaging of CXCR4 receptor expression in human cancer xenografts with [64Cu]AMD3100 positron emission tomography.</a> Cancer Res. 70, 3935-3944 (2010).</li><li>De Silva, R. A., Peyre, K., Pullambhatla, M., Fox, J. J., Pomper, M. G., Nimmagadda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+CXCR4+expression+in+human+cancer+xenografts:+evaluation+of+monocyclam+64Cu-AMD3465.">Imaging CXCR4 expression in human cancer xenografts: evaluation of monocyclam 64Cu-AMD3465.</a> J Nucl Med. 52, 986-993 (2011).</li><li>Hatse, S., Princen, K., Liekens, S., Vermeire, K., De Clercq, E., Schols, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+CXCL12AF647+as+a+novel+probe+for+nonradioactive+CXCL12/CXCR4+cellular+interaction+studies.">Fluorescent CXCL12AF647 as a novel probe for nonradioactive CXCL12/CXCR4 cellular interaction studies.</a> Cytometry A. 61, 178-188 (2004).</li><li>Wootten, D., Christopoulos, A., Sexton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+paradigms+in+GPCR+allostery:+implications+for+drug+discovery.">Emerging paradigms in GPCR allostery: implications for drug discovery.</a> Nat Rev Drug Discov. 12, 630-644 (2013).</li><li>Louis, K. S., Siegel, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+viability+analysis+using+trypan+blue:+manual+and+automated+methods.">Cell viability analysis using trypan blue: manual and automated methods.</a> Methods Mol Biol. 740, 7-12 (2011).</li><li>Perry, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc:+a+review+of+its+use+in+the+management+of+CCR5-tropic+HIV-1+infection.">Maraviroc: a review of its use in the management of CCR5-tropic HIV-1 infection.</a> Drugs. 70, 1189-1213 (2010).</li><li>Moyle, G., DeJesus, E., Boffito, M., Wong, R. S., Gibney, C., Badel, K., MacFarland, R., Calandra, G., Bridger, G., Becker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proof+of+activity+with+AMD11070,+an+orally+bioavailable+inhibitor+of+CXCR4-tropic+HIV+type+1.">Proof of activity with AMD11070, an orally bioavailable inhibitor of CXCR4-tropic HIV type 1.</a> Clin Infect Dis. 48, 798-805 (2009).</li><li>Balabanian, K., Lagane, B., Infantino, S., Chow, K. Y., Harriague, J., Moepps, B., Arenzana-Seisdedos, F., Thelen, M., Bachelerie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemokine+SDF-1/CXCL12+binds+to+and+signals+through+the+orphan+receptor+RDC1+in+T+lymphocytes.">The chemokine SDF-1/CXCL12 binds to and signals through the orphan receptor RDC1 in T lymphocytes.</a> J Biol Chem. 280, 35760-35766 (2005).</li><li>Burns, J. M., Summers, B. C., Wang, Y., Melikian, A., Berahovich, R., Miao, Z., Penfold, M. E., Sunshine, M. J., Littman, D. R., Kuo, C. J., Wei, K., McMaster, B. E., Wright, K., Howard, M. C., Schall, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chemokine+receptor+for+SDF-1+and+I-TAC+involved+in+cell+survival,+cell+adhesion,+and+tumor+development.">A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.</a> J Exp Med. 203, 2201-2213 (2006).</li><li>Stoddart, L. A., Kilpatrick, L. E., Briddon, S. J., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+pharmacology+of+G+protein-coupled+receptors+with+fluorescent+ligands.">Probing the pharmacology of G protein-coupled receptors with fluorescent ligands.</a> Neuropharmacology. 98, 48-57 (2015).</li><li>Vernall, A. J., Hill, S. J., Kellam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolving+small-molecule+fluorescent-conjugate+toolbox+for+Class+A+GPCRs.">The evolving small-molecule fluorescent-conjugate toolbox for Class A GPCRs.</a> Br J Pharmacol. 171, 1073-1084 (2014).</li><li>Stoddart, L. A., White, C. W., Nguyen, K., Hill, S. J., Pfleger, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-+and+bioluminescence-based+approaches+to+study+GPCR+ligand+binding.">Fluorescence- and bioluminescence-based approaches to study GPCR ligand binding.</a> Br J Pharmacol. 173, 3028-3037 (2016).</li></ol>

Compared to other types of binding assays (i.e., saturation binding and kinetic binding experiments), competition&#160;binding assays are best suited for screening purposes. Indeed, they allow evaluation of large batches of unlabeled compounds, for instance small molecules, by scoring their capability to interfere with the binding of a fixed amount of a labeled receptor ligand. Compounds that bind to other receptor sites than the labeled ligand might remain undetected in the assay. Although the competition bindi...

G protein-coupled receptors (GPCRs) are cell surface proteins that can be activated by extracellular ligands (e.g., peptides, protein hormones, amines), thereby regulating many physiological and developmental processes1. When an agonist occupies its GPCR binding pocket, the induced conformational change in the receptor protein promotes the binding of intracellular receptor-associated heterotrimeric G proteins, consisting of G&#945;-GDP and G&#946;&#947; subunits. The subsequent exchange of GTP for GDP on the G&#945; subunit results in the dissociation of the G protein subunits (G&#945;-GTP and G&#946;&#947;) that, in turn, will further initiate downstream signaling pathways2,3. When the G&#945;-GTP becomes hydrolyzed, re-association of the G&#945;-GDP and G&#946;&#947; subunits will convert the G protein back into its resting state3,4. Distinct types of G proteins exist (Gs, Gi/o, Gq, G12/13), which are categorized based on sequence similarity with the G&#945; subunit5. All of these G proteins induce defined intracellular signaling pathways that underlie the biological response to receptor activation. Subsequent to receptor activation, GPCR kinases (GRKs) phosphorylate the intracellular tail of GPCRs, thereby promoting interaction with &#946;-arrestins. This process leads to the termination of G protein signaling, receptor desensitization and internalization6. &#946;-arrestins are also part of multi-molecular complexes that trigger signaling cascades independent of G protein signaling7.
GPCRs are amongst the most validated molecular targets for therapeutic intervention, as deregulated GPCR-mediated signaling, for instance due to gain-of-function mutations in the receptor gene or receptor overexpression, contributes to the etiology of many human diseases8. Therefore, GPCRs represent one of the most important classes of drug targets investigated by the pharmaceutical industry8,9,10. A notable example of a clinically relevant GPCR is the CXC chemokine receptor 4 (CXCR4), which can be activated by a sole natural ligand, the CXC chemokine ligand 12 (CXCL12)11. Due to its established role as a major co-receptor for human immunodeficiency virus 1 (HIV-1) entry and infection in cluster of differentiation 4 (CD4) positive T-lymphocytes12, CXCR4 was first investigated as an antiviral drug target. CXCL12-CXCR4 interaction in the bone marrow further regulates the retention and homing of stem and progenitor cells13. Also, given its involvement in many aspects of cancer biology (e.g., tumor cell survival, metastasis, tumor-related angiogenesis)14 and several other human diseases (e.g., inflammatory diseases)15, CXCR4 raised significant interest as a promising target for drug discovery. AMD3100, a small molecule that specifically targets CXCR4, was initially discovered as an anti-HIV drug candidate16 and is still one of the most potent CXCR4 antagonists described to date17. Its development as an antiviral drug was, however, discontinued18. Currently this molecule is used as a stem cell mobilization agent during the treatment of multiple myeloma and lymphoma patients18. Several other chemically unrelated small molecules and biologics that inhibit CXCR4 function with varying potency have been described19.
Receptor binding methods are valuable tools in pharmacology that allow the identification of compounds (e.g., small molecules) that directly interact with the GPCR of interest. In order to perform binding studies, there is no need for prior knowledge concerning the intracellular signaling properties or functionality of a given GPCR. Although this can be considered to be an advantage, it implies that compounds for which receptor binding can be demonstrated need to be further characterized by evaluating their potential agonistic or antagonistic activity. This activity can be evaluated using pharmacological or biological assays related to the GPCR under study. Dependent on their activity profile, receptor binding molecules might then potentially evolve to become novel lead compounds for investigation in pre-clinical and clinical studies. Molecules that specifically bind to a receptor with high affinity can also serve as scaffolds to generate therapeutic or diagnostic tools, for instance by radiolabeling them for noninvasive in vivo imaging of tumor cells20, or as potential vehicles for targeted delivery of therapeutics21. In case of CXCR4, in vivo imaging of tumor cells has already been demonstrated using mouse models wherein labeled CXCR4-targeting molecules allowed the visualization of human cancer xenografts20,22,23.
In this report, we describe a detailed protocol for a competition binding assay that enables the identification of small molecules and biologics that directly interfere with agonist (CXCL12) binding to CXCR4. The basic principle of the assay is the competition between a fixed amount of fluorescently labeled ligand (CXCL12AF647, see Table of Materials and Reagents) and unlabeled compounds for binding to the receptor protein17,24. The specific fluorescent signal from labeled ligand bound to single cells expressing CXCR4 is then analyzed by flow cytometry. This fluorescent signal will be reduced when unlabeled small molecules disrupt the interaction between CXCL12AF647 and CXCR4. The assay uses non-manipulated living cells that endogenously express CXCR4 (i.e., Jurkat cells). Hence, no cell membrane preparation is required, which makes the assay convenient, fast and compatible with increased throughput. Since a fluorescently labeled ligand is used, radioactivity is avoided.
Because CXCL12 is the natural agonist for CXCR4, small molecule compounds that interfere with CXCL12AF647 binding in the assay are likely to interact with the orthosteric receptor binding site (i.e., the binding site occupied by the natural agonist). Molecules that would interact with receptor binding sites topographically distinct from the orthosteric binding site remain undetected,&#160;if they do not influence binding of CXCL12. For instance, positive and negative allosteric modulators, an important and emerging category of GPCR targeting molecules acting on allosteric binding sites25, will potentially not be picked up with this assay. In addition, whether the compounds identified with this binding assay function as receptor antagonists or as agonists cannot be derived. Investigation of the identified compounds in additional pharmacological or functional receptor-related assays will thus be required. These assays might include (a combination of) cellular fluorescence- or luminescence-based assays for the detection of second messengers (e.g., Ca2+, cyclic adenosine monophosphate (cAMP)), phenotypic or biological assays and &#946;-arrestin recruitment assays, the choice of which depends on the specific signaling properties of the GPCR under study. Hence, the competitive binding assay described herein mainly serves as an initial screening assay that needs to be complemented with other cell-based assays to enable an in-depth characterization of compounds with receptor binding potency.

<table border="0" cellpadding="0" cellspacing="0" width="1217">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSCanto II</td>
			<td style="width:247px;">Becton Dickinson</td>
			<td style="width:281px;">Not applicable</td>
			<td style="width:304px;">Flow cytometry device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSDIVA Software</td>
			<td style="width:247px;"></td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSArray</td>
			<td style="width:247px;">Becton Dickinson</td>
			<td style="width:281px;">Not applicable</td>
			<td>Flow cytometry device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">BD FACSArray System Software</td>
			<td style="width:247px;"></td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graphpad Prism</td>
			<td style="width:247px;">Graphpad</td>
			<td style="width:281px;"></td>
			<td>software package used for nonlinear regression analysis in Figure 2 and Figure 3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:387px;">FlowJo</td>
			<td style="width:247px;">FlowJo is now a wholly owned subsidiary of BD.</td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Vi-CELL</td>
			<td style="width:247px;">Beckman Coulter</td>
			<td style="width:281px;">Not applicable</td>
			<td>cell viability analyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Sigma 3-18 KS</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">Not applicable</td>
			<td>centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">AMD3100</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">A5602-5mg</td>
			<td>specific CXCR4 antagonist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Maraviroc</td>
			<td style="width:247px;">Pfizer</td>
			<td style="width:281px;"></td>
			<td>antiretroviral drug, CCR5 antagonist, available for research at Selleckchem (cat#S2003), Sigma (cat#PZ0002)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">h-SDF1a (AF647)</td>
			<td style="width:247px;">ALMAC</td>
			<td style="width:281px;">CAF-11-B-01</td>
			<td>fluorescently labeled CXCL12, CXCL12AF647</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">A1933-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">HBSS (10x), calcium, magnesium, no phenol red</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">14065-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">HEPES (1M)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">15630-056</td>
			<td style="width:304px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:247px;">Gibco (Life Technologies)</td>
			<td style="width:281px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Jurkat cells</td>
			<td style="width:247px;">ATCC</td>
			<td style="width:281px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Reagent reservoir PP</td>
			<td style="width:247px;">Sigma</td>
			<td style="width:281px;">BR703411</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rapid flow filter: 0.2 &#181;m aPES</td>
			<td style="width:247px;">Thermo Scientific</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Sterilin microtiter plate, 96-well, U bottom, clear</td>
			<td style="width:247px;">Thermo Scientific</td>
			<td style="width:281px;">611U96</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Falcon tubes, 50ml</td>
			<td style="width:247px;">Greiner Bio-One</td>
			<td style="width:281px;">227 261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:387px;">Tissue culture flask (T75)</td>
			<td style="width:247px;">Corning</td>
			<td style="width:281px;">353024</td>
			<td></td>
		</tr>
	</tbody>
</table>

a flow cytometry-based assay to identify compounds that disrupt binding of fluorescently-labeled cxc chemokine ligand 12 to cxc chemokine receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

The general workflow of the binding assay is presented in Figure 1A. An illustration of the type of flow cytometry data obtained for different sample types in a standard experiment (i.e., negative control, positive control, and experimental sample) is depicted in Figure 1B, and a possible plate layout to perform the assay in a 96-well plate format is given in Figure 1C. Incubation of Jurkat ...

Watch this Scientific Journal Video about A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4 at JoVE.c

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

The overall goal of the cell-based Assay is to identify molecules that bind to a G protein coupled receptor. Based on their capability to compete with a fluorescently-labeled ligand for binding to the receptor. Competition binding assay can support G-protein coupled receptor drug discovery.As we will demonstrate with this protocol for the chemokine receptor, CXC4. The main advantage of this method is that living cells are used to analyze receptive binding instead of memory preparations. And that the use of radioactivity label probes is avoided.Though this method has initially been set up to study binding to the chemokine receptor CXCR4, it can also be developed for other GPCR for which fluorescently-labeled ligands exist. To begin, dispense 100 microliters of compound solution into a clear 96 well rounded bottom plate, according to a predefined experimental layout. Add 50 microliters of cell suspension from a reagent reservoir into the 96 well plate using a multi-channel pipette.Incubate the plate for 15 minutes at room temperature in the dark. Following incubation, add 50 microliters of fluorescently-labeled 100 nanograms per milliliter CXCL12 from a similar reagent reservoir to the wells of the 96 well-plate. Incubate for 30 minutes at room temperature in the dark.Next, centrifuge the 96 well plate at 400 times gravity for five minutes at room temperature. Remove the supernatant from the pelleted cells by flipping over the plate. Dry the plate on a tissue.Add 200 microliters of fresh SA buffer from a reagent reservoir to the wells using a multi-channel pipette. Immediately proceed to centrifuge the plate again, for five minutes at 400 times gravity at room temperature. Again remove the supernatant, by flipping over the plate and then drying it on a tissue.Finally, gently resuspend the cell pellet in 200 microliters of 1%paraformaldehyde dissolved in PBS. This step will fix the cells. To analyze the samples by flow cytometry, start up the device and open the corresponding software.Select the cellular parameters to be visualized in a dot block format, as forward scatter area, side scatter area, and in a histogram view, the fluoro four detection channel. Choose one sample. For instance a negative control sample, to perform gating of a defined homogenous cell population based on the forward scatter and side scatter parameters.Adapt the settings for loading the cells into the flow cytometry device. Select three mixes before injection. Select automatic injection of 100 microliters of fixated cells, and use a sample flow rate of 1.5 microliters per second.Run the sample by selecting acquire data. The forward scatter and side scatter parameters for this sample, as well as the fluoro four detection will now appear on the screen. Now, select the software gating tool.Based on the forward scatter and side scatter dot plot visualization, predefine a homogenous and viable cell population by gating. Select to analyze 20, 000 events per sample. This means that for each sample, 20, 000 cells that fall within a predefined gates, will eventually be analyzed.Data acquisition will continue until this number of events is reached. Start the run by selecting first, the wells to be analyzed. Then selects run wells.The flow cytometry device will now analyze these samples one by one, by recording the mean fluorescence intensity for each sample. Which corresponds to the mean fluorescent signal from the 20, 000 cells that fall within the predefined gate. Finally, perform data analysis as described in the protocol.Following pre-incubation of jurkat cells with high concentrations of AMD3100, unestablished and specific CXCR4 antagonist. The mean fluorescence intensity that reflects binding of labeled CXCL12 is almost entirely reduced to background levels. This demonstrates the highly specific binding of CXCL12 to CXCR4 expressed on jurkat cells.Compounds that are unable to compete with labeled CXCL12 for binding at the CXCR4 receptor, like the CCR5 antagonist Maraviroc, will not affect the mean fluorescence intensity. In contrast, highly active compounds will do this in a dose-dependent manner. After watching the video, you should have a good understanding of how to perform a competition receptive binding assay using living cells and flow cytometry.

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