Name: in vitro imaging and quantification of the drug targeting efficiency of fluorescently labeled gnrh analogues
Uploaded: 2017-03-21T15:00:00.000+00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues at JoVE.com

The project was supported by National Research Fund OTKA (K 104045).

1. Preparation of Cell Cultures and Reagents
<ol>
	<li>Maintain the cell cultures in the manufacturer&#39;s recommended medium, supplemented with 10% (v/v) fetal bovine serum and antibiotics (called complete medium). Keep the cell culturing flask in a humidified, 5% CO2 atmosphere incubator at 37 &#176;C. Follow the proliferation and confluency of cells by inverted microscope (using 10X phase contrast objective).</li>
	<li>When cells reach adequate confluency, remove the medium, and wash the culture with 2-3 mL, sterile phosphate-buffered saline (PBS). Remove the PBS and add 0.5 mL, sterile 0.25% trypsin-EDTA solution to the cell culture and incubate at 37 &#176;C until cells detach (approximately 10 min).</li>
	<li>Suspend the cells in 3-4 mL sterile complete medium to stop trypsin and transfer them into a sterile centrifuge tube. Centrifuge the cells at 150 x g for 4 min at room temperature (RT). Discard the supernatant carefully and suspend the pellet in 2-3 mL sterile complete medium.</li>
	<li>Take out 100 &#181;L of the cell suspension and mix with 100 &#181;L 0.4% (m/V) trypan blue solution, to stain the dead cells. Load 10 &#181;L of this mixture into a hemocytometer and determine the number of viable cells by inverted microscope.</li>
	<li>Dilute the required amount of cell suspension prepared in step 1.3 to 10 mL with sterile complete medium, containing 4 x 104 cell/mL.</li>
	<li>Add 250 &#181;L of cell suspension (prepared in step 1.5) per well (104 cell/well) into seven wells of the first glass bottom 8-well microscopic slide. Use this slide in the GnRH uptake method.</li>
	<li>Add 250 &#181;L of cell suspension (prepared in step 1.5) per well (104 cell/well) into five wells of the second 8-well microscopic slide. This slide will be used in the GnRH-I-R expression method.</li>
	<li>Add 1 mL of cell suspension (prepared in step 1.5) per well (4 x 104 cell/well) into seven wells of a 12-well plate. This plate will be used in the GnRH quantification method.</li>
	<li>Let the cells adhere on the two microscopic slides and the plate. Incubate them in a humidified, 5% CO2 atmosphere incubator at 37 &#176;C for 48 h.</li>
	<li>After the incubation check the cells by inverted microscope (using 10X phase contrast objective). If the cells are healthy and attached, continue with the following steps.</li>
	<li>Prepare 10 mM GnRH-FITC stock solution in dimethyl sulfoxide (DMSO) from each of the three conjugates18. (Use these dilution concentrations: 16.43 &#181;g/&#181;L [D-Lys6(FITC)]-GnRH-I, 16.97 &#181;g/&#181;L [D-Lys6(FITC)]-GnRH-II and 16.47 &#181;g/&#181;L [Lys8(FITC)]-GnRH-III) Keep these stock solutions in a dark place at RT and use them up within a few weeks.</li>
	<li>Dilute 1.7 &#181;L each of the three 10 mM GnRH-FITC stock solutions in 1.7 mL complete medium. Shake these solutions. (These are the 10 &#181;M GnRH-FITC treating media.) Dilute 150 &#181;L each of the three 10 &#181;M GnRH-FITC treating medium in 1.35 mL complete medium. Shake these solutions as well (these are the 1 &#181;M GnRH-FITC treating medium). 
	NOTE: All GnRH-FITC containing treating medium should be protected from light and used up within a few h.</li>
	<li>Preheat the six GnRH-FITC treating medium (prepared in step 1.12) and 4 mL complete medium to 37 &#176;C.</li>
	<li>Dilute 3 &#181;L of 5 mM fluorescent probe solution (nuclear counterstain mentioned in the Materials List) in 3 mL PBS to 5 &#181;M. This solution should be protected from light, keep it at RT and use it up within a few hours.</li>
</ol>
2. Confocal Laser Scanning Microscopy (CLSM)
<ol>
	<li>GnRH uptake method
	<ol>
		<li>Take the first microscopic slide prepared in step 1.6 and pipette out the complete medium from each of the seven well. Add 250 &#181;L of preheated complete medium into the first well. Use this as a negative control. Add 250 &#181;L of the preheated GnRH-FITC treating media (from 1.13) to each of the next six wells (3 wells with 1 &#181;M and 3 with 10 &#181;M). Incubate the slide in a humidified, 5% CO2 atmosphere incubator at 37 &#176;C for 5 h.</li>
		<li>Pipette out the medium from each well and wash the cells with 250 &#181;L PBS. Add 250 &#181;L of fixing solution (10% neutral buffered formalin) into each well and incubate the slide at RT, for 10 min.</li>
		<li>Pipette out the fixing solution from each well and wash the cells with 250 &#181;L PBS. Add 250 &#181;L of PBS containing 5 &#181;M fluorescent probe solution prepared in step 1.14 into each well and incubate the slide at RT, for 10 min.</li>
		<li>Pipette out the fluorescent probe solution from each well, and wash the cells twice with 250 &#181;L PBS carefully. Add 3-4 drops of mounting medium into each well, finally. Keep the slide in dark at RT, until the imaging.</li>
	</ol>
	</li>
	<li>GnRH-I-R expression method
	<ol>
		<li>Take the second 8-well microscopic slide prepared in step 1.7 and pipette out the complete medium from each of the five wells.</li>
		<li>Add 250 &#181;L of preheated complete medium into the first well, use this as negative control. Add 250 &#181;L of preheated complete medium into the second well too, and use this to examine GnRH-I-R expression before the GnRH treatment.</li>
		<li>Add 250 &#181;L each of the three preheated 10 &#181;M GnRH-FITC treating media into the next three wells. Use these wells to pretreat the cells with GnRH-FITC conjugates before examining the GnRH-I-R expression. Incubate the slide in a humidified, 5% CO2 atmosphere incubator at 37 &#176;C for 1 h.</li>
		<li>Pipette out the treating medium from each of the three wells which will be used to examine GnRH-I-R expression after the GnRH treatment and wash these wells with 250 &#181;L of preheated complete medium. Add 250 &#181;L of preheated complete medium into each of the three wells. Incubate the slide in a humidified, 5% CO2 atmosphere incubator at 37 &#176;C for 1 h.</li>
		<li>Pipette out the medium from each of the five wells and wash the cells with 250 &#181;L PBS. Add 250 &#181;L of fixing solution into each well and incubate the slide at RT, for 10 min.</li>
		<li>Pipette out the fixing solution from each well and wash the cells with 250 &#181;L of PBS. Add 250 &#181;L of PBS containing 5% bovine serum albumin (BSA) blocking solution into each well and incubate the slide at RT, for 1 h.</li>
		<li>Dilute 10 &#181;L of GnRH-I-R primary antibody in 1 mL PBS (1:100 ratio). Keep it at RT and use it up within a few hours.</li>
		<li>Pipette out the BSA blocking solution from each well except the negative control (first well). Wash the cells with 250 &#181;L PBS (except the negative control well). Add 250 &#181;L of PBS containing GnRH-I-R primary antibody prepared in step 2.2.7 into each well (except the negative control). Incubate the slide at RT, for 1 h, in a dark place.</li>
		<li>Dilute 2.5 &#181;L of Alexa 546 labeled secondary antibody in 1.25 mL PBS (1:500 ratio). 
		NOTE: This solution should be protected from light, keep it at RT and use it up within a few hours.</li>
		<li>Pipette out solutions from each of the five wells and wash the cells with 250 &#181;L PBS. Add 250 &#181;L of PBS containing AF 546 labeled secondary antibody prepared in step 2.2.9 into each well. Incubate the slide at RT, for 1 h in dark.</li>
		<li>Pipette out the solution from each well and wash the cells with 250 &#181;L PBS. Add 250 &#181;L of PBS containing 5 &#181;M fluorescent probe solution prepared in step 1.14 into each well and incubate the slide for 10 min, at RT.</li>
		<li>Pipette out the fluorescent probe solution from each well and wash the cells twice with 250 &#181;L PBS carefully. Add 3-4 drops of mounting media into each well, afterwards. Keep the slide in dark at RT until the microscopic imaging.</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Image the cells by inverted confocal laser scanning microscope (using 63X oil immersion objective)
		<ol>
			<li>Use the following excitation/emission wavelengths. GnRH conjugates: 488/514 nm, Alexa 546 labeled antibody: 488/546 nm (use only for the GnRH-I-R expression method), Draq5 fluorescent probe: 633/680 nm.</li>
			<li>Set up the imaging parameters using the negative control cells. Use these parameters to image treated cells (Figure 3). Re-adjust imaging parameters on each microscopic slide at the beginning of the analysis. Fine-tune and export images with the software provided by the manufacturer if necessary.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Fluorescence-activated Cell Sorting (FACS)
<ol>
	<li>GnRH quantification method
	<ol>
		<li>Take the plate prepared in step 1.8 and pipette out the complete medium from each of the seven wells. Add 1 mL of preheated complete medium into the first well. Use this as a negative control. Add 1 mL each of the six preheated treating media (3 wells with 1 &#181;M and 3 wells with 10 &#181;M) into the next six wells. Incubate the plate in a humidified, 5% CO2 atmosphere incubator at 37 &#176;C for 5 h.</li>
		<li>Pipette out the medium from each well. Wash the cells twice with 2 mL PBS carefully. Add 500 &#181;L of trypsin-EDTA solution into each wells. Incubate the plate at 37 &#176;C until cells detach (approximately 10 min).</li>
		<li>Add 1 mL of complete medium into each well to stop trypsin. Shake the plate. Suspend the cells gently with a pipette, and transfer the suspension from each well into FACS tubes. Centrifuge the tubes at 150 x g for 4 min, at 4 &#176;C.</li>
		<li>Pour out the supernatants carefully, with one move. Add 500 &#181;L of ice-cold PBS into each FACS tube and gently re-suspend the cells. Keep the tubes on ice until the end of the experiment.</li>
	</ol>
	</li>
	<li>Analysis and calculation
	<ol>
		<li>Analyze the cells by flow cytometry. For excitation, use 488 nm argon laser wavelength and for detection use 530 nm wavelength. Set up the flow cytometer parameters by running the negative control cells. Use these parameters to analyze treated cells (Figure 4A). Evaluate the data with the manufacturer&#39;s software, determine median fluorescent intensity (MFI) values, and calculate the relative MFI values.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Experiments described herein use selectively labeled GnRH analogues for screening adherent cell cultures in vitro. Confocal microscopy and flow cytometry methods are suitable for tracking and quantifying the cellular uptake of these GnRH-FITC conjugates in a time and concentration dependent manner. These experiments have the following critical steps: 1) maintain a sterile, healthy cell culture; 2) GnRH peptides must be of high quality; 3) reasonable concentrations and incubation times must be followed; ...

Peptide based targeted drug delivery systems have become a fast developing and promising area in cancer therapy over the past few years1,2. Human gonadotropin releasing hormone receptor type I (GnRH-I-R) is primarily located in the pituitary gland but is also present in several other tissues which are responsible for self-reproduction3. GnRH-I-R is also expressed in a number of cancer tissues, related or unrelated to the reproductive system4,5. The high expression of GnRH-I-R on several malignant tumor cells compared with healthy tissues provides an opportunity for targeted therapy5,6.
Many gonadotropin-releasing hormone (GnRH) analogues have been developed in the last few decades, which could be used as targeting moieties7,8,9. These peptides are able to deliver anticancer agents with high selectivity into malignant tumor cells which over-express GnRH-I-R6. Several GnRH conjugated anti-tumor drugs with higher selectivity and better efficiency than the corresponding unconjugated free drug have been reported7,8,9.
Previous publications about GnRH peptides and their receptors reported that the GnRH-I-R can assume various conformations which have different selectivity for GnRH analogues10. The highly variable GnRH-I-R has complex and various signaling pathways are endowed with different activity against their natural and artificial ligands11. These facts make investigation of GnRH-based systems challenging. On the other hand, they possess promising therapeutic potential. Several experiments with radiolabeled GnRH peptides were previously reported12,13,14,15, but experiments in which fluorescently labeled GnRH analogues were used are still limited. While radioactive labeling offers high sensitivity, fluorescent labeling has several other advantages, for example the easier handling, and the ability to counterstain with different fluorophores. Three common GnRH analogues which have successfully been used for drug delivery are the [D-Lys6]-GnRH-I, [D-Lys6]-GnRH-II and GnRH-III, but the effectiveness of these peptides as targeting moieties is rarely compared16,17. On the other hand, results from separate experiments in which different cancer cells and GnRH analogues were used is diverse.
Based on these considerations, we focused on the tumor targeting and drug delivery potential of these GnRH peptides, and thereby synthesized and characterized the [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III peptide conjugates18. These analogues are selectively labeled with FITC on the side chain of their Lys or D-Lys (peptide-FITC ratio 1:1 at each conjugate). The idea was that the selective fluorescent labeling can offer novel information about these peptides, and allows their good tracking and reliable quantification. These conjugates have safe handling and reliable detectability, which make it easier to compare their tumor targeting efficiency, and the screening of numerous types of malignant tumor cells. We hope that up-to date experiments with these peptide conjugates could contribute to the development of novel cancer targeting GnRH-drug conjugates, and help to identify new therapeutic targets as well.
The present manuscript demonstrates some well reproducible and fast experiments with GnRH-FITC conjugates. The cell surface expression of GnRH-R is a determinative condition regarding GnRH uptake, therefore we simultaneously investigated the cell surface level of GnRH-I-R on the tested cell lines. We visualized the GnRH-I-R and GnRH-FITC conjugates by confocal laser scanning microscopy (CLSM) and quantified the cellular uptake of GnRH-FITC conjugates using fluorescence-activated cell sorting (FACS).

<table><tbody><tr><td>EBC-1</td><td>JCRB Cell Bank</td><td>JCRB0820</td><td>Human lung squamous cell carcinoma</td></tr><tr><td>BxPC-3</td><td>ATCC</td><td>CRL-1687</td><td>Human pancreatic adenocarcinoma</td></tr><tr><td>Detroit-562</td><td>ATCC</td><td>CCL-138</td><td>Human pharyngeal carcinoma</td></tr><tr><td>RPMI-1640 Medium with L-glutamine</td><td>Lonza</td><td>BE12-702F</td><td>Culture medium for BxPC-3 cells</td></tr><tr><td>Eagle&#039;s Minimum Essential Medium</td><td>Lonza</td><td>BE12-611F</td><td>Culture medium for EBC-1 cells, and complemented with 0.1% sodium pyruvate for Detroit-562 cells</td></tr><tr><td>Fetal Bovine Serum</td><td>Euro Clone</td><td>ECS0180L</td><td>Complements the culture medium</td></tr><tr><td>MycoZap Plus-CL&amp;nbsp;</td><td>Lonza</td><td>VZA-2012</td><td>Complements the culture medium (antibiotics)</td></tr><tr><td>Standard Line Cell Culture Flasks</td><td>VWR</td><td>10062-872</td><td>Provide consistent, sterile growth environment for cells</td></tr><tr><td>Trypsin-EDTA Mixture&amp;nbsp;</td><td>Lonza</td><td>BE17-161E</td><td>Remove attached cells</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Lonza</td><td>BE17-516F</td><td>Solvent</td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418-100ML</td><td>Solvent</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250061</td><td>Used to counting cells</td></tr><tr><td>[D-Lys&lt;sup&gt;6&lt;/sup&gt;(FITC)]-GnRH-I</td><td>made by us</td><td>-</td><td>Purity &amp;ge;98% (HPLC)</td></tr><tr><td>[D-Lys&lt;sup&gt;6&lt;/sup&gt;(FITC)]-GnRH-II</td><td>made by us</td><td>-</td><td>Purity &amp;ge;98% (HPLC)</td></tr><tr><td>[Lys&lt;sup&gt;8&lt;/sup&gt;(FITC)]-GnRH-III</td><td>made by us</td><td>-</td><td>Purity &amp;ge;98% (HPLC)</td></tr><tr><td>glass bottom 8-well microscopic slide</td><td>Ibidi</td><td>80826</td><td>Use in CLSM experiment</td></tr><tr><td>Corning Costar cell culture plate, 12 well</td><td>Sigma-Aldrich</td><td>CLS3513-50EA</td><td>Use in FACS experiment</td></tr><tr><td>Fixing solution (10% neutral buffered formalin)</td><td>Bio-Optica</td><td>01V60P</td><td>Fix adherent cells</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906-10G</td><td>Prevent the nonspecific binding of the antibodies</td></tr><tr><td>GnRHR Antibody</td><td>Proteintech</td><td>19950-1-AP</td><td>Immunocytochemistry reagent, bind to the human type-I GnRH receptors</td></tr><tr><td>Secondary Antibody, Alexa Fluor 546 conjugate</td><td>Thermo Fisher Scientific</td><td>A11035</td><td>Immunocytochemistry reagent, bind to the primary antibody</td></tr><tr><td>Fluorescent probe solution (Draq5)</td><td>Thermo Fisher Scientific</td><td>62254</td><td>Counterstain nuclei</td></tr><tr><td>Fluoromount Aqueous Mounting Medium</td><td>Sigma-Aldrich</td><td>F4680-25ML</td><td>Use in CLSM experiment, preserving fluorescence</td></tr><tr><td>Inverted microscope</td><td>Elektro-Optika Ltd.&amp;nbsp;</td><td>Alpha XDS-2T</td><td>Check the phenotype and confluency of cell cultures</td></tr><tr><td>Inverted confocal laser scanning microscope</td><td>Zeiss</td><td>LSM-710</td><td>Use in CLSM experiment</td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td>BD FACSCalibur</td><td>Use in FACS experiment</td></tr></tbody></table>

in vitro imaging and quantification of the drug targeting efficiency of fluorescently labeled gnrh analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues&#39; cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

Images obtained by confocal laser scanning microscopy (CLSM) offer spectacular information about the uptake of GnRH-FITC conjugates on the given cell culture in a time and concentration dependent manner. In parallel with these GnRH-FITC conjugates, the presence of the GnRH-I-R on the cell surface is also verifiable by the CLSM experiment. Furthermore, by using a far-red DNA staining fluorescent probe, it is possible to counterstain the nuclei of cells besides GnRH and GnRH-I-R. However, w...

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In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

in-vitro-imaging-quantification-drug-targeting-efficiency

Research

JoVE Journal

Cancer Research

7.6K Views.  Semmelweis University.  Selectief gemerkte fluorescerende GnRH-I, -II en -III derivaten betrouwbare middelen voor het opsporen en kwantificeren van de cellulaire opname. Dit manuscript introduceert experimenten te visualiseren, kwantificeren en te vergelijken de opname efficiëntie van deze GnRH conjugaten in diverse cellijnen. 

Video: In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Chemistry

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Bioengineering

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Biochemistry

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Medicine

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Biology

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

Neuroscience

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

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