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 means of flow cytometry. A description of the flow cytometry procedure and reagents to determine receptor expression levels at the cell surface is, however, not within the scope of this protocol, but has been described previously17.</li>
	<li>Let Jurkat cells grow in suspension until they reach 80 - 85% confluency. Before passaging the cells to a novel flask, allow all reagents to reach room temperature (RT).</li>
	<li>Add 20 mL of fresh complete growth medium (RPMI-1640 medium, 10% fetal bovine serum (FBS), 2 mM glutamine) to a novel T75 culture flask.</li>
	<li>Add 5 mL of Jurkat cell suspension from the original T75 flask (containing 25 mL of cell suspension) to the novel T75 culture flask. Incubate at 37 &#176;C and 5% CO2 in a humidified incubator.</li>
</ol>
2. Preparation of CXCL12, Assay Buffer, and Jurkat Cells for the Competition Binding Assay.
<ol>
	<li>Prepare a stock solution of CXCL12AF647 (20 &#181;g/mL; see Table of Materials and Reagents) by dissolving the lyophilized reagent (stored at -80 &#176;C, in the dark) in ultrapure water supplemented with 0.01% (volume/volume) of Polysorbate 20. Store single use aliquots from this stock solution at -80 &#176;C, protected from light.</li>
	<li>Prepare assay buffer by adding 40 mL HEPES (1 M, 20 mM final concentration) to 200 mL Hank&#39;s Balanced Salt Solution (HBSS, 10x, without phenol red and without sodium bicarbonate, 1x final concentration). Add ultrapure water to obtain a final volume of 2 L. Add 4 g (0.2% weight/volume) bovine serum albumin (BSA), and dissolve the BSA via magnetic stirring. Finally, adjust the pH to 7.4 (use NaOH for this) and filter the solution through 0.2 &#181;m pores (see Table of Materials and Reagents) using a vacuum manifold. 
	NOTE: This assay buffer will be used in all further steps of the protocol.</li>
	<li>Count the number and the viability of the cells. For this, take a sample of the cell suspension and dilute it in phosphate buffered saline (PBS). 
	NOTE: We routinely use an automated cell viability analyzer (see Table of Materials and Reagents) capable of counting cell suspensions at varying concentrations. For cell counting, dilute 0.5 mL of cell suspension in 1.5 mL PBS (other dilutions, e.g., 0.1 mL in 1.9 mL PBS, are also possible). The use of this method, which is based on the trypan blue dye exclusion method, has been described previously26. Several other devices are commercially available for counting cell number and viability that should work equally well.</li>
	<li>Collect the desired number of cells (i.e., ~24 x 106 cells to run the assay with one complete 96-well plate) in a sterile 50 mL tube by centrifugation (see Table of Materials and Reagents for the type of centrifuge used) at 400 x g for 5 min at RT.</li>
	<li>Gently pour off the supernatant without disturbing the cell pellet. Add fresh assay buffer (e.g., 20 mL) and resuspend the cells by gently pipetting up and down.</li>
	<li>Centrifuge the cells again at 400 x g for 5 min at RT.</li>
	<li>Pour off the supernatant again and resuspend the cell pellet in fresh assay buffer to obtain a density of 5 x 106 cells/mL.</li>
</ol>
3. Competition Binding Assay
NOTE: The actual competition binding assay is performed at RT and can be performed under non-sterile conditions.
<ol>
	<li>Dilute the compounds under investigation in assay buffer (see 2.2) to obtain the desired concentration. Either prepare a fixed concentration of compound for initial screening (e.g., 10 &#181;M final concentration) or, alternatively, a serial dilution series of concentrations for more detailed characterization of the compounds (e.g., a 1/3, 1/4 or 1/5 dilution series starting at 1 &#181;M, final concentration). Keep in mind that the compound solution will ultimately become 2x diluted in the assay; therefore, prepare a 2x concentrated solution.</li>
	<li>Dispense 100 &#181;L of compound solution (2x concentrated) into a clear 96-well round bottom plate (see Table of Materials and Reagents) according to a pre-defined experimental lay out (e.g., Figure 1C). 
	NOTE: At this stage, negative and positive control samples are included in the assay. In the negative control sample, 100 &#181;L of assay buffer is added instead of compound to the wells of the 96-well plate. For the positive control sample, assay buffer is also added during this step. See also Figure 1C for a typical experimental layout in which a dilution series of several compounds is tested.</li>
	<li>Add 50 &#181;L of cell suspension (see 2.7; 0.25 x 106 cells) from a reagent reservoir into the 96-well plate using a multichannel pipette. Incubate the plate for 15 min at RT in the dark.</li>
	<li>Add 50 &#181;L of fluorescently labeled CXCL12 (i.e., 100 ng/mL of CXCL12AF647 in assay buffer, 4x concentrated, 25 ng/mL final concentration) from a similar reagent reservoir to the wells of the 96-well plate. Incubate for 30 min at RT in the dark. 
	NOTE: For the negative control samples, add assay buffer instead. Hence, the fluorescent signal detected in the negative control samples will correspond to the autofluorescent background signal (Figure 1A). For the positive control samples, add 50 &#181;L/well of CXCL12AF647. The positive control samples will yield the maximal fluorescence signal detected, since no potential inhibition by pre-incubation with compounds was included (Figure 1A).</li>
	<li>Centrifuge the 96-well plate at 400 x g for 5 min at RT. Remove the supernatant from the pelleted cells by flipping over the plate. Dry the plate on a tissue.</li>
	<li>Add 200 &#181;L of fresh assay buffer from a reagent reservoir to the wells using a multichannel pipette. Immediately proceed.</li>
	<li>Centrifuge the plate again for 5 min at 400 x g at RT. Remove the supernatant by flipping over the plate and again dry it on tissue.</li>
	<li>Gently resuspend the cell pellet in 200 &#181;L of 1% paraformaldehyde dissolved in PBS. This step will fix&#160;the cells.</li>
	<li>Continue the protocol immediately with the quantification of the fluorescence by flow cytometry.</li>
</ol>
4. Analysis of the samples by flow cytometry
CXCL12AF647 stained and fixated cells are now ready to be analyzed using flow cytometry. Several types of flow cytometers can be used, but they need to be equipped with the correct laser (i.e., a red laser, excitation range ~630 nm) for excitation and suitable filters for fluorophore detection (emission filters ~660 nm). They need to be capable of handling samples in a 96-well plate format. Examples of suitable flow cytometry devices are given in the Table of Materials and Reagents.
<ol>
	<li>Start up the device and open the corresponding software (see Table of Materials and Reagents).</li>
	<li>Select the following cellular parameters to be visualized in a dot blot format: forward scatter (FSC), side scatter (SSC) and the fluorophore (CXCL12AF647) detection channel. 
	NOTE: With the FSC parameter, cells are discriminated based on their size, since the detected light absorption is proportional to the cell&#39;s diameter. The SSC parameter, measuring light scattering at a 90&#176; angle, provides information about the granularity of the cells.</li>
	<li>Choose one sample (e.g., a negative control sample) to perform gating of a defined homogenous cell population based on the FSC and SSC parameters.
	<ol>
		<li>Select automatic injection of ~100 &#181;L of fixated cells from this negative control sample into the flow cytometer.Select the option &#34;mixing&#34; before injection and use a sample flow rate of 1.5 &#181;L/s.</li>
		<li>Run this sample by selecting &#34;Acquire Data.&#34;&#160;The FSC and SSC parameters for this sample will now appear on the screen.</li>
		<li>Select the software&#39;s gating tool. Based on the FSC and SSC dot blot visualization, pre-define a homogenous and viable cell population by gating. To do so, create a polygon (using the software&#39;s gating tool) that includes the homogenously distributed single cells (&#34;events&#34;) based on these two dimensions. 
		NOTE: The gating procedure aims to define a homogenous and viable cell population that will be used for further analysis. Gating relies on the assumption that the majority of viable cells will form a homogenous cell population based on the FSC and SSC parameters. By performing this step, cellular debris, dead cells, and cell aggregates can largely be excluded from further analysis. An illustration of the gating process is given in Figure 1B.</li>
	</ol>
	</li>
	<li>Select to analyze 20,000 &#34;events&#34; (i.e., single cells) per sample. 
	NOTE: This means that for each sample, 20,000 cells that fall within the pre-defined gate will eventually be analyzed. Data acquisition for each sample will continue until this number of events is analyzed.</li>
	<li>Start the run (select &#34;Record Data&#34;). The flow cytometry device will now analyze all samples one-by-one by recording the mean fluorescence intensity (MFI) for each sample. This MFI corresponds to the mean fluorescent signal corresponding to the 20,000 cells that fall within the pre-defined gate.</li>
</ol>
5. Data analysis
Use the MFI obtained for each sample to perform all further calculations. Analysis of the flow cytometry data can be performed by several commercially available software packages (see Table of Materials and Reagents).
<ol>
	<li>To determine the percentage inhibition of the fluorescent binding signal, as a result of compound pre-incubation, apply the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/57271/57271eq1.jpg" /> 
	Where: 
	MFIExp = the MFI of the experimental (= compound-treated) sample 
	MFIPC = the MFI of the positive control 
	MFINC = the MFI of the negative control</li>
	<li>To determine the IC50 value of a compound (i.e., the concentration of compound that can reduce the fluorescent binding signal by 50%), apply the following formula: 
	<img alt="Equation 2" src="/files/ftp_upload/57271/57271eq2.jpg" /> 
	Where: 
	Logconc.2 = the log of a concentration of compound that results in less than 50% inhibition of the difference between the MFI value of the PC and NC 
	Logconc.1 = the log of a concentration of compound that results in more than 50% inhibition of the difference between the MFI value of the PC and NC 
	NOTE: Alternatively, in case of highly active compounds, a dose-response curve covering several logs of magnitude can be generated based on the MFI corresponding to each tested concentration of compound. By applying non-linear regression curve fitting using appropriate software (see Table of Materials and Reagents), IC50 values can then be deduced from the generated curves. An example of this analysis approach is shown in Figure 3.</li>
</ol>

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The authors have nothing to disclose.

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 binding experiment described herein focuses on the chemokine receptor CXCR4 in particular, it can likely serve as a blueprint for the design of competition binding assays for other GPCRs as well. For instance, the CXC chemokine receptor 7 (CXCR7) was described as an alternative receptor for CXCL12, capable of binding CXCL12 with high affinity29,30. Hence, the same labeled chemokine (CXCL12AF647) as used for the described CXCR4 competition binding assay can be used to set up CXCR7 binding experiments. Because CXCR4 and CXCR7 share CXCL12 as a common ligand, it would be important to work with cellular models in which only one of both receptors is expressed at the cell surface, in order to evaluate specific receptor binding. Our previous work has demonstrated that Jurkat cells do not show endogenous expression of CXCR717. Thus, in the assay described here for CXCR4, interference with binding to CXCR7 is ruled out.
Several key features of the competition binding assay allow it to be used as a fast, initial, screening tool to identify small molecules interfering with agonist binding to CXCR4. One of the assay&#39;s key benefits is the use of whole, living cells expressing the receptor of interest. This omits the need for labor-intensive cell membrane preparations. Also, the low level of background (auto)fluorescence detected with Jurkat cells is beneficial. Together with the large signal window obtained upon the addition of the defined amount of fluorescently labeled probe, it contributes to a favorable signal to background ratio. This facilitates the detection of potential binding inhibition by unlabeled compounds. In case this binding assay is performed with other CXCR4 expressing cells, we recommend to always evaluate these parameters (background fluorescence, signal window) upfront as they might differ from cell line to cell line. The fact that Jurkat cells endogenously express CXCR4 at a consistent level over long periods, makes it a convenient cellular model. The same consistent level of receptor expression might be obtained with stably transfected cell clones. In contrast, we do not recommend the use of transiently transfected cells because this will likely result into a much broader distribution of fluorescence intensities, fewer responsive cells in the assay and less consistency between experiments. Since no CXCR4 negative Jurkat cells exist, specificity of receptor binding to these cells was demonstrated by pre-incubation with well-established specific receptor antagonists (AMD3100 and AMD11070). These antagonists are structurally different from the labeled ligand. This is important because, when using the unlabeled variant of the ligand as competitor, both labeled and unlabeled ligand might also compete for binding to non-specific binding sites potentially present at the cell surface.
Apart from the cellular host, several other aspects of the assay described here need to be taken into account before starting to build a similar assay for other GPCRs. Fluorescently labeled probes that target GPCRs have been developed as alternatives for radioactive probes, but are currently only available for a limited number of GPCRs and they are relatively expensive. Moreover, modification of natural receptor ligands with fluorophores might alter the binding properties as compared to the unlabeled parent molecule, especially in case of very small ligands (e.g., amines) or small peptides31,32. For instance, in case of small ligands, a linker is generally required to separate the pharmacophore from the fluorophore to allow access to the receptor&#39;s orthosteric binding site31. Also, the stability of the receptor-fluorescent probe complex needs to be taken into account. In our experience, after 90 - 120 minutes (i.e., the time needed to run two consecutive 96-well plates), the mean fluorescent intensity for the positive control wells typically is reduced by ~5% compared to the average of all positive control wells on the plate, with occasional outliers between 5 and 10%.
Also, the intrinsic activity of the labeled ligand is of importance. Whereas receptor antagonists display no intrinsic activity on the receptor, labeled agonists likely induce receptor activation and, in consequence, receptor internalization. This compromises the reversible nature of the ligand-receptor interaction33. To minimize possible issues with receptor internalization, incubation&#160;with the labeled ligand (in case of a labeled agonist) need to be limited in time and should preferentially be performed at room temperature or lower. The fact that the procedure is performed at room temperature makes the assay also convenient and more compatible with screening efforts in plate format.&#160;Finally, a sufficient number of cells per sample (e.g., 250,000 per well of a 96-well plate) should be used in the assay to ultimately retain a sufficient number of cells (20,000 cells, &#34;single events,&#34; based on the gating of the whole cell population; see Figure 1) for flow cytometry analysis. Using less cells would make it more difficult to consistently reach this number of analyzed cells, which might compromise the reliability of the read out. Using less cells per sample (per well) would also increase the time interval needed to analyze the sample by flow cytometry, and thus increases the overall time to analyze a whole 96-well plate. This&#160;compromises increased throughput. Due to the abovementioned reason, future miniaturization of the assay might thus turn out not to be a trivial task.
Taken together, a competition binding assay that is primarily used in our lab as a screening tool to identify small molecules that can compete with a fixed amount of labeled CXCL12 for binding to CXCR4 has been described in detail. These binding studies are of interest as they are a relatively fast and simple method to identify compounds capable of receptor binding. Although the identification of compounds with binding capacity is already valuable in itself, this type of assay does not provide information about the activity of the identified compounds. Hence, it remains necessary to further validate and characterize their potential agonistic or antagonistic activity in other pharmacological and functional receptor assays. Of interest, in the case of CXCR4 it has been shown that receptor antagonists that show increased activity in the binding assay (i.e., that generate low IC50 values for binding inhibition) perform relatively better in other functional CXCR4-related assays as well17.

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 cells, which endogenously express the chemokine receptor CXCR4 (i.e., the GPCR of interest in this assay), with increasing amounts of fluorescently labeled CXCL12 (i.e., CXCL12AF647) resulted into increased levels of fluorescence (Figure 2), reflecting increased levels of ligand binding to CXCR4. To demonstrate the receptor-specificity of CXCL12AF647 binding, a well-established and receptor-specific CXCR4 antagonist (the small molecule AMD3100) was used. Jurkat cells were first pre-incubated with a 1/5 dilution series of AMD3100 ranging from 0.064 ng/mL to 1,000 ng/mL, followed by an incubation with a final concentration of 25 ng/mL CXCL12AF647 (i.e., the fixed amount of labeled chemokine routinely used in the assay). Then, Jurkat cells were further processed as described in the protocol. The fluorescent signal (the mean fluorescent intensity, MFI) after addition of the fixed amount of CXCL12AF647 to the pre-incubated Jurkat cells, was obtained by flow cytometry analysis for all samples. This fluorescent signal could almost be entirely inhibited in case Jurkat cells were pre-treated with the highest concentration of AMD3100 (1,000 ng/mL). Under this condition, the inhibited level of fluorescence was only slightly above the background level corresponding to the level of autofluorescence for Jurkat cells. In conclusion, applying 25 ng/mL (final concentration) of CXCL12AF647 in the binding assay generated a large fluorescent signal window (when compared to the negative control) with a minimal residual level of non-specific receptor binding (Figure 3A).
This binding assay has primarily been used to screen for compounds disrupting ligand binding in panels of unlabeled compounds at a single fixed concentration, or to study the dose-dependent effect of active compounds. In the latter case, the goal is to obtain IC50 values (i.e., the concentration of compound that inhibits the binding signal by 50%), which reflect the binding potency of the compounds. Figure 3 illustrates how flow cytometry data obtained with this binding assay can be used to determine the inhibitory potency (in terms of IC50) of small molecules disrupting the binding of CXCL12 to CXCR4. Based on the dose-dependent inhibition of CXCL12AF647 binding by AMD3100, the IC50 value was determined here by applying non-linear regression analysis. In this example, the calculated IC50 value for AMD3100 corresponds to 18.12 ng/mL. Because in screening campaigns, the majority of randomly selected compounds are expected not to inhibit the binding of CXCL12AF647 to CXCR4+ Jurkat cells, an example of an inactive compound was also included in this study. Here, the small molecule maraviroc, which exhibits potent anti-HIV-1 activity by acting as a specific antagonist for the CC chemokine receptor 5 (CCR5)27, was tested in the binding assay. No inhibition of the maximal fluorescent binding signal was detected after pre-incubating Jurkat cells with maravoric, even at the highest concentration tested (100 ng/mL; Figure 3B). In the same experiment, a serial dilution series of AMD11070, another small molecule related to AMD3100, but with improved pharmacokinetic properties28, was included. Unlike maraviroc, a 1/5 dilution series from AMD11070 ranging from 0.0064 to 100 ng/mL clearly and dose-dependently inhibited CXCL12AF647 binding (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57271/57271fig1.jpg" /> 
Figure 1: Overview of the workflow and illustration of the type of data obtained. (A). The main steps of the protocol are schematized for the negative and positive control samples, and compound-treated samples. Samples without compound pre-incubation and without addition of fluorescently labeled chemokine (CXCL12AF647) are included to determine the background (auto)fluorescence (negative control samples). Samples without compound pre-incubation, but with addition of a fixed amount of CXCL12AF647 are used to determine the maximal binding signal in the assay (positive control samples). In compound-treated samples, cells are pre-incubated with compound before addition of a fixed amount of CXCL12AF647. (B). The mean fluorescent binding signal is determined by flow cytometry analysis of Jurkat cells. From all events (i.e., Jurkat cells) analyzed (left panel) only the fluorescent signal from a gated subpopulation of cells is used for further analysis (middle panel). Pre-incubation of cells with small molecules (e.g., AMD3100) inhibits the binding of the fluorescently labeled receptor ligand and this will reduce the maximal binding signal (right panel, shown in a histogram representation). (C). A possible plate layout when performing the competitive binding assay in a 96-well plate format. In this case, six different compounds (cpd 1-&#160; 6) are tested in a 1/5 serial dilution series (in duplicate) ranging from 1,000 nM down to 0.32 nM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57271/57271fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57271/57271fig2.jpg" /> 
Figure 2: Binding of CXCL12AF647 to Jurkat cells. Jurkat cells were incubated with increasing amounts of CXCL12AF647, in absence of pre-incubation with compound. The mean fluorescence intensity (MFI, expressed in arbitrary light units) &#177; SD is shown (n = 2). Curve fitting was performed using nonlinear regression using a one-site total binding model following the equation
<img alt="Equation 3" src="/files/ftp_upload/57271/57271eq3.jpg" />
with Bmax (maximum specific binding) = 18,345; Kd = 92.8 ng/mL; NS (the slope of nonspecific binding) = 2.139; and Background = 332.6. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57271/57271fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57271/57271fig3.jpg" /> 
Figure 3: Inhibition of the binding of CXCL12AF647 by active and inactive compounds. (A) Dose-dependent inhibition of CXCL12AF647 binding by pre-incubating cells with increasing concentrations of AMD3100, before addition of 25 ng/mL CXCL12AF647. Note that the maximal inhibited mean fluorescence intensity (MFI; expressed in arbitrary light units) is still slightly above the background (auto)fluorescence obtained for Jurkat cells (Mean &#177; SD (n = 3)). Curve fitting was performed using non-linear regression with a variable slope (four parameters) according to
<img alt="Equation 4" src="/files/ftp_upload/57271/57271eq4.jpg" />
Here the HillSlope is 1.313 and the calculated IC50 is 18.12 ng/mL. (B). Dose-dependent effect of AMD11070 and maraviroc on CXCL12AF647 binding to CXCR4+ Jurkat cells (MFI &#177; SD (n = 3)). Curve fitting for the AMD11070 response was performed similarly with a HillSlope of 1.458 and a calculated IC50 value of 0.9052 ng/mL. For the maraviroc response, no fitting was performed given the lack of activity of this compound. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57271/57271fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

a-flow-cytometry-based-assay-to-identify-compounds-that-disrupt

Nanyang Technological University

Research

JoVE Journal

Biology

13.6K Views.  KU Leuven. 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).

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

Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys loop, the glutamate-gated and the P2X receptor channels2. In each case binding of transmitter leads to the opening of a pore through which ions flow down their electrochemical gradients. Many ligand-gated channels are also permeable to calcium ions3, 4, which have downstream signaling roles5 (e.g. gene regulation) that may exceed the duration of channel opening. Thus ligand-gated channels can signal over broad time scales ranging from a few milliseconds to days. Given these important roles it is necessary to understand how ligand-gated ion channels themselves are regulated by proteins, and how these proteins may tune signaling. Recent studies suggest that many, if not all, channels may be part of protein signaling complexes6. In this article we explain how to identify the proteins that bind to the C-terminal aspects of the P2X2 receptor cytosolic domain.
P2X receptors are ATP-gated cation channels and consist of seven subunits (P2X1-P2X7). P2X receptors are widely expressed in the brain, where they mediate excitatory synaptic transmission and presynaptic facilitation of neurotransmitter release7. P2X receptors are found in excitable and non-excitable cells and mediate key roles in neuronal signaling, inflammation and cardiovascular function8. P2X2 receptors are abundant in the nervous system9 and are the focus of this study. Each P2X subunit is thought to possess two membrane spanning segments (TM1 &amp; TM2) separated by an extracellular region7 and intracellular N and C termini (Fig 1a)7. P2X subunits10 (P2X1-P2X7) show 30-50% sequence homology at the amino acid level11. P2X receptors contain only three subunits, which is the simplest stoichiometry among ionotropic receptors. The P2X2 C-terminus consists of 120 amino acids (Fig 1b) and contains several protein docking consensus sites, supporting the hypothesis that P2X2 receptor may be part of signaling complexes. However, although several functions have been attributed to the C-terminus of P2X2 receptors9 no study has described the molecular partners that couple to the intracellular side of this protein via the full length C-terminus. In this methods paper we describe a proteomic approach to identify the proteins which interact with the full length C-terminus of P2X2 receptors.

We describe a simple protocol to identify brain proteins that bind to the full length C terminus of ATP-gated P2X2 receptors. The extension and systematic application of this approach to all P2X receptors is expected to lead to a better understanding of P2X receptor signaling.

Ligand-gated ion channels underlie synaptic communication in the nervous system1. In mammals there are three families of ligand-gated channels: the cys ...

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biochemical Reconstitution of Steroid Receptor&#x2022;Hsp90 Protein  Complexes and Reactivation of Ligand Binding

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, including transcription factors (e.g. nuclear receptors, p53) and protein kinases (e.g. Src, Raf, Akt kinase) involved in cell cycling, tumorigenesis, apoptosis, and multiple eukaryotic signaling pathways 1,2. Of these many 'client' proteins for hsp90, the assembly of steroid receptor&bull;hsp90 complexes is the best defined (Figure 1). We present here an adaptable glucocorticoid receptor (GR) immunoprecipitation assay and in vitro GR&bull;hsp90 reconstitution method that may be readily used to probe eukaryotic hsp90 functional activity, hsp90-mediated steroid receptor ligand binding, and molecular chaperone cofactor requirements. For example, this assay can be used to test hsp90 cofactor requirements and the effects of adding exogenous compounds to the reconstitution process. 
The GR has been a particularly useful system for studying hsp90 because the receptor must be bound to hsp90 to have an open ligand binding cleft that is accessible to steroid 3. Endogenous, unliganded GR is present in the cytoplasm of mammalian cells noncovalently bound to hsp90. As found in the endogenous GR&bull;hsp90 heterocomplex, the GR ligand binding cleft is open and capable of binding steroid. If hsp90 dissociates from the GR or if its function is inhibited, the receptor is unable to bind steroid and requires reconstitution of the GR&bull;hsp90 heterocomplex before steroid binding activity is restored 4 . GR can be immunoprecipitated from cell cytosol using a monoclonal antibody, and proteins such as hsp90 complexed to the GR can be assayed by western blot. Steroid binding activity of the immunoprecipitated GR can be determined by incubating the immunopellet with [3H]steroid. 
Previous experiments have shown hsp90-mediated opening of the GR ligand binding cleft requires hsp70, a second molecular chaperone also essential for eukaryotic cell viability. Biochemical activity of hsp90 and hsp70 are catalyzed by co-chaperone proteins Hop, hsp40, and p23 5. A multiprotein chaperone machinery containing hsp90, hsp70, Hop, and hsp40 are endogenously present in eukaryotic cell cytoplasm, and reticulocyte lysate provides a chaperone-rich protein source 6. 
In the method presented, GR is immunoadsorbed from cell cytosol and stripped of the endogenous hsp90/hsp70 chaperone machinery using mild salt conditions. The salt-stripped GR is then incubated with reticulocyte lysate, ATP, and K+, which results in the reconstitution of the GR&bull;hsp90 heterocomplex and reactivation of steroid binding activity 7. This method can be utilized to test the effects of various chaperone cofactors, novel proteins, and experimental hsp90 or GR inhibitors in order to determine their functional significance on hsp90-mediated steroid binding 8-11.

An in vitro method for preparing functional glucocorticoid receptor (GR)&#x2022;hsp90 protein complexes from purified proteins and cellular lysates is described. The method utilizes immunoadsorption of recombinant GR followed by salt-stripping and protein complex reconstitution. The importance of cofactors and buffer conditions are discussed, as are potential method applications.

Hsp90 is an essential and highly abundant molecular chaperone protein that has been found to regulate more than 150 eukaryotic signaling proteins, ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...