an in vitro technique to study glutamate receptor trafficking in hippocampal neurons

An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons

To begin this procedure, transfer the coverslips cell-side up to a paraffin film-covered tray to save reagents and facilitate manipulation. Save and maintain conditioned media at 37 degrees Celsius for incubation and washing steps.
Incubate the cells with primary antibodies diluted in conditioned media at room temperature for 15 minutes. After this, use a vacuum pipette to carefully aspirate off the antibody-containing media and wash the cells three times with conditioned media.
If studying surface versus intracellular receptor expression, fix the cells with paraformaldehyde after this step as outlined in the text protocol. Maintain the cells in conditioned media without antibodies and return them to the incubator at 37 degrees Celsius to allow for internalization. If studying internalization process, fix the cells with paraformaldehyde after this step, as outlined in the text protocol.
To block the epitopes on the primary antibody attached to the surface-expressed receptors that have not been internalized, incubate cells with unconjugated Fab anti-IgG antibody fragments diluted in conditioned media for 20 minutes at room temperature. After this, wash the cells three times with conditioned media. Incubate the washed wells with conditioned media containing 80 micromolar dynasore at 37 degrees Celsius to allow for recycling of internalized receptors.
After finishing the modifications on live cells, wash the cells once with PBS+. Add a solution of 4% paraformaldehyde and 4% sucrose in PBS to the cells, and incubate in a fume hood at room temperature for 7 to 8 minutes to fix the cells. Then, wash the cells three times with regular PBS. Add a solution of 10% normal goat serum in PBS to the cells, and incubate at room temperature for 30 minutes to block nonspecific binding sites.
Next, incubate the cells with fluorescently-tagged secondary antibody diluted in 3% NGS in PBS at room temperature for one hour to label primary antibody-labeled receptors. After this, wash the cells three times with PBS.
To begin labeling the intracellular receptors, add a solution of 0.25% Triton-X in PBS and incubate at room temperature for 5 to 10 minutes to permeabilize the cells. Then, block with 10% NGS at room temperature for 30 minutes. If studying surface versus total, incubate the cells with the same primary antibody used previously, diluted in 3% NGS in PBS at room temperature for one hour to label the intracellular receptors.
Next, wash the cells three times with PBS. Label with second fluorescently-tagged secondary antibody diluted in 3% NGS in PBS at room temperature for one hour. After this, wash the cells three times with PBS. First, gently place the coverslips, cell-side down, on 12 to 15 microliters of the appropriate mounting media to mount the cells. Then, image the cells on the appropriate confocal microscope, and analyze the cells as outlined in the text protocol.

an-vitro-technique-to-study-glutamate-receptor-trafficking

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Characterization and Functional Analysis

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1. Preparation Before Labeling
<ol>
	<li>Preparation and maintenance of primary hippocampal cultures&#8203;
	<ol>
		<li>Prepare primary hippocampal cultures at a density of 150,000 cells plated on poly-D-lysine-coated (0.1 mg/mL) 18 mm cover glasses. Excellent guides for dissociated neuronal culture preparation are available. 
		NOTE: If required, the cultures may be treated with cytosine arabinoside (Ara-C, 10 &#956;M from days in vitro 1 [DIV1]) to avoid glial proliferation in the preparation. 
		NOTE: Alternative coating reagents such as fibronectin (1 mg/mL) or laminin (5 &#956;g/mL) may be used instead of poly-D-lysine.</li>
		<li>Maintain cultures in a cell incubator at 37 &#176;C and 5% CO2 in 2 mL/well of neurobasal media supplemented with B27 and 2 mM L-glutamine. 
		NOTE: Substitutes for L-glutamine (e.g., Glutamax) can be used, if desired.</li>
		<li>On weekly-basis, remove half the volume of media and replace with the same volume of supplemented neurobasal media.</li>
	</ol>
	</li>
	<li>OPTIONAL: Transfection of mutated and/or epitope-tagged receptors 
	NOTE: Neurons should be transfected at least 3&#8211;4 days prior to the analysis time point to allow for receptor expression. The use of young neurons (DIV6&#8211;9) results in better transfection efficiency than older (DIV15&#8211;20) neurons, but a sufficient number of transfected cells (&#62;20) can be achieved regardless of the DIV employed.
	<ol>
		<li>For each well of a 12-well plate, dilute 1.5 &#956;g of plasmid containing the construct of interest in 100 &#956;L of fresh neurobasal media without B27 or glutamine supplementation in a microcentrifuge tube and mix by vortexing quickly. 
		NOTE: For successful transfection, it is critical that the neurobasal media used is as fresh as possible, ideally less than 1 week after bottle opening.</li>
		<li>In a second microcentrifuge tube, mix 1 &#956;L of an appropriate lipofection reagent in 100 &#956;L of fresh neurobasal media and mix gently. 
		NOTE: Do not vortex the lipofection reagent mixture. Use of fresh lipofection reagents can improve transfection efficiency.</li>
		<li>Incubate the tubes for 5 min at room temperature (RT).</li>
		<li>Add the lipofection reagent mixture dropwise to the DNA mixture, mix gently, and incubate for 20 min at RT.</li>
		<li>Adjust the volume of media in each well to 1 mL of conditioned media.</li>
		<li>Add the lipofection reagent -DNA mixture dropwise to the well.</li>
		<li>Return cells to the incubator and allow at least 3&#8211;4 days for protein expression. 
		NOTE: For the purposes of the internalization and recycling protocols outlined below, hippocampal neurons were transfected at DIV11&#8211;12 with constructs expressing GluN2B tagged with green fluorescent protein (GFP) in the extracellular domain (GFP-GluN2B) and imaged at DIV15&#8211;16.</li>
	</ol>
	</li>
	<li>OPTIONAL: Incubation of cells with drugs (chronically or acutely) in the conditioned media until fixation. 
	&#8203;NOTE: For acute treatment, begin treating cells before labeling. Depending on the drug treatment protocol used, cells can be maintained in drug-containing media during section 2. In our example, DIV21 cells were subject to a chemically induced long-term potentiation (cLTP) protocol to increase surface-expressed &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR).
	<ol>
		<li>Exchange conditioned media for extracellular solution (ECS).</li>
		<li>Treat cells with 300 &#956;M glycine in ECS for 3 min at RT. As a control, treat a sister coverslip with ECS (without glycine).</li>
		<li>Wash cells 3x with 37 &#176;C ECS and return the cells in ECS (without glycine) to the cell incubator for 20 min prior to continuing with section 2. 
		NOTE: ECS (in mM): 150 NaCl, 2 CaCl2, 5 KCl, 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 30 Glucose, 0.001 tetrodotoxin (TTX), 0.01 strychnine, and 0.03 picrotoxin at pH 7.4.</li>
	</ol>
	</li>
</ol>
2. Live Labeling of Surface-expressed Receptors
<ol>
	<li>Prepare coverslips for labeling&#8203;
	<ol>
		<li>To save reagents and facilitate manipulation, transfer coverslips cell side up to a paraffin film-covered tray. 
		&#8203;NOTE: It is critical to never let the samples dry out.</li>
		<li>Save and maintain conditioned media at 37 &#176;C for incubation and washing steps. 
		NOTE: For an 18 mm coverslip, incubation with 75&#8211;100 &#956;L of media for antibody labeling and 120&#8211;150 &#956;L for internalization/recycling are recommended.</li>
	</ol>
	</li>
	<li>Labeling of surface receptors with primary antibody&#8203;
	<ol>
		<li>Incubate cells with primary antibody diluted in conditioned media for 15 min at RT. 
		NOTE: For GFP-tagged receptors, rabbit anti-GFP antibody at a dilution of 1:1000 was used. For endogenous GluA1, mouse anti-GluA1 at a 1:200 dilution was used.</li>
		<li>Carefully aspirate off the antibody-containing media using a vacuum pipette and wash cells three times with conditioned media. 
		NOTE: If conditioned media is unavailable, all washing steps may be performed using PBS+ [phosphate buffered saline (PBS) containing 1 mM MgCl2 and 0.1 mM CaCl2]. Manual aspiration using a micropipette may be performed if gentle vacuum aspiration is not available.</li>
	</ol>
	</li>
</ol>
3. Surface Expression (Figure 1)
<ol>
	<li>Secondary antibody labeling of surface-expressed receptors
	<ol>
		<li>Wash once with PBS+.</li>
		<li>Fix cells by incubating with 4% paraformaldehyde (PFA) and 4% sucrose in PBS for 7&#8211;8 min. 
		NOTE: Unlike other fixation methods such as methanol incubation, PFA does not permeabilize the plasma membrane and is therefore suitable for surface-expression analysis. For optimal results, use freshly prepared PFA. Short-term storage of PFA at 4 &#176;C or long-term (up 30 days) storage at -20 &#176;C is permissive for adequate fixation. 
		CAUTION: PFA is a known carcinogen. Use proper personal protective equipment and a safety hood when handling.</li>
		<li>Wash cells three times with regular PBS. 
		NOTE: Alternatively, 0.1 M glycine can be used for washing PFA instead of PBS, as glycine will quench any remaining fixative that may increase the background in the preparation.</li>
		<li>Block nonspecific binding sites by incubating with 10% normal goat serum (NGS) in PBS for 30 min at RT. 
		NOTE: Blocking time can be extended without adverse effects on labeling.</li>
		<li>Incubate with fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT to label primary antibody-labeled receptors (i.e., surface-expressed). 
		NOTE: In these examples, a 1:500 dilution of Alexa 555-conjugated secondary antibodies:goat anti-rabbit for GFP-labeled receptors and goat anti-mouse for GluA1 was used.</li>
		<li>Wash cells with PBS 3x.</li>
	</ol>
	</li>
	<li>Labeling of intracellular receptors
	<ol>
		<li>Permeabilize cells with 0.25% Triton X-100 in PBS for 5&#8211;10 min at RT. 
		NOTE: To check that the initial round of antibody labeling occupies all surface epitopes, this permeabilization step can be skipped in a sister culture. In this case, no signal for intracellular receptors should be obtained. Additionally, to check that no internal receptors have been labeled in the previous section 2 (i.e., showing the integrity of the plasma membranes in culture), the permeabilization step can be skipped in a sister culture, and a primary antibody against an intracellular protein (e.g., postsynaptic density protein 95 [PSD-95] or microtubule-associated protein 2 [MAP2]) can be utilized in step 2.2.1. No signal should be obtained from this primary under these conditions. In this case, a rabbit anti-PSD-95 antibody (1:500) was used.</li>
		<li>Block with 10% NGS in PBS for 30 min at RT.</li>
		<li>Label intracellular receptors by incubating permeabilized cells with the same primary antibody used in section 2.2 diluted in 3% NGS in PBS for 1 h at RT. 
		NOTE: The antibody dilution for labeling intracellular receptors may be different than that required for labeling surface-expressed receptors. In the example of GluA1, the same antibody dilution (1:200) was used.</li>
		<li>Wash cells 3x with PBS.</li>
		<li>Label with second fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT. 
		NOTE: In these examples, a 1:500 dilution of goat anti-mouse Alexa 647-conjugated secondary antibody (for GluA1) was used.</li>
		<li>Wash cells 3x with PBS.</li>
	</ol>
	</li>
</ol>
4. Internalization (Figure 2)
<ol>
	<li>Internalization of antibody-labeled surface receptors&#8203;
	<ol>
		<li>After labeling of surface-expressed receptors and antibody washing (section 2.2), maintain cells in conditioned media without antibody and return them to the incubator (37 &#176;C) to allow for internalization. 
		&#8203;NOTE: For N-methyl-D-aspartate (NMDA) receptors, 30 min for internalization is recommended. As a control, a sister culture may be maintained with conditioned media at 4 &#176;C during the internalization process. Minimal receptor internalization should occur under these conditions.</li>
	</ol>
	</li>
	<li>Labeling of surface receptors
	<ol>
		<li>Wash cells once with PBS+.</li>
		<li>Fix cells with 4% PFA and 4% sucrose in PBS for 7&#8211;8 min. 
		CAUTION: Use proper personal protective equipment and a safety hood when handling PFA.</li>
		<li>Wash cells 3x with regular PBS.</li>
		<li>Block with 10% NGS in PBS for 30 min at RT to prevent nonspecific binding.</li>
		<li>Incubate samples with fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT to label primary antibody-labeled receptors (i.e., surface-expressed receptors which were not internalized). 
		NOTE: For this example, Alexa 555-conjugated goat anti-rabbit secondary antibody (1:500) was used for labeling.</li>
		<li>Wash cells 3x with PBS.</li>
	</ol>
	</li>
	<li>Labeling of internalized receptors
	<ol>
		<li>Permeabilize cells with 0.25% Triton X-100 in PBS for 5&#8211;10 min.</li>
		<li>Block nonspecific binding by incubation with 10% NGS in PBS for 30 min at RT.</li>
		<li>Incubate samples with fluorescently tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT to label internalized antibody-labeled receptors. 
		NOTE: For this example, Alexa 647-conjugated goat anti-rabbit secondary antibody (1:500) is used for labeling.</li>
		<li>Wash cells 3x with PBS.</li>
	</ol>
	</li>
</ol>
5. Recycling (Figure 3)
<ol>
	<li>Internalization of antibody-labeled surface receptors&#8203;
	<ol>
		<li>After labeling of surface-expressed receptors and antibody washing (section 2.2), maintain cells in conditioned media without antibody and return them to the incubator (37 &#176;C) to allow for internalization. 
		&#8203;NOTE: For NMDA receptors, 30 min for internalization is recommended.</li>
	</ol>
	</li>
	<li>Blocking of stable surface expressed receptors
	<ol>
		<li>To block the epitopes on the primary antibody attached to surface-expressed receptors that have not been internalized, incubate cells with unconjugated Fab anti-IgG (H+L) antibody fragments (against the primary used in section 2.2) diluted in conditioned media (20 &#956;g/mL) for 20 min at RT. This treatment prevents future interaction with secondary antibodies. 
		NOTE: For this example, Goat anti-rabbit Fab fragments were used. 
		NOTE: Control experiment: to ensure that complete blocking of surface-expressed receptors has occurred, sister coverslips can be incubated with and without Fab. Cultures should be fixed immediately after Fab treatment, and both cultures are incubated with Alexa 555-conjugated secondary antibody. No Alexa 555 signal in the Fab-incubated cells indicates proper antibody blocking.</li>
		<li>Wash cells 3x with conditioned media.</li>
		<li>Incubate cells with conditioned media containing 80 &#956;M dynasore to prevent further internalization and return cells to the incubator (37 &#176;C) to allow for recycling of internalized receptors. Dynasore is a GTPase inhibitor that inhibits dynamin and therefore prevents internalization. 
		NOTE: 45 min for NMDAR recycling is recommended. Note that Dynasore exclusively blocks the dynamin-dependent internalization process (e.g., NMDARs internalization). However, internalization of other synaptic protein (dynamin-independent) can still occur in the presence of Dynasore.</li>
	</ol>
	</li>
	<li>Labeling of recycled receptors
	<ol>
		<li>Wash cells once with PBS+.</li>
		<li>Fix cells with 4% PFA and 4% sucrose in PBS for 7&#8211;8 min. 
		CAUTION: Use proper personal protective equipment and a safety hood when handling PFA.</li>
		<li>Wash cells 3x with PBS.</li>
		<li>Block with 10% NGS in PBS for 30 min at RT to prevent nonspecific binding.</li>
		<li>Label cells with first fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT. 
		NOTE: For this example, Alexa 555-conjugated goat anti-rabbit antibody (1:500) was used for labeling.</li>
		<li>Wash cells 3x with PBS. 
		NOTE: Longer washes with PBS (5&#8211;10 min) may help to reduce background in the preparation.</li>
	</ol>
	</li>
	<li>Labeling of internalized receptors
	<ol>
		<li>Permeabilize cells with 0.25% Triton X-100 in PBS for 5&#8211;10 min.</li>
		<li>Block with 10% NGS in PBS for 30 min at RT.</li>
		<li>Label with second fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT. 
		NOTE: For this example, Alexa 647-conjugated goat anti-rabbit antibody (1:500) was used for labeling.</li>
		<li>Wash cells 3x with PBS.</li>
	</ol>
	</li>
</ol>
6. Mounting and Imaging of Samples
<ol>
	<li>Mount cells by gently placing the coverslips cell side down on 12&#8211;15 &#956;L of the appropriate mounting media. 
	NOTE: Aspiration of excess mounting media will improve the quality of images.</li>
	<li>Image cells on an appropriate confocal microscope. 
	NOTE: It is recommended to image a z-stack at 60x magnification with 0.35 &#956;m steps, encompassing the entire thickness of the neuron.</li>
</ol>
7. Time Considerations
<ol>
	<li>This is a long protocol that can be stopped at several points. If desired, perform blocking and primary antibody incubation steps overnight at 4 &#176;C in a humid chamber.</li>
	<li>Alternatively, if desired, use a microwave tissue processor to vastly speed up post-fixation incubation times. For all steps, use 150 W at 30 &#176;C for &#34;On&#34; settings.&#8203;
	<ol>
		<li>To block, run the processor at 2 min &#34;On,&#34; 1 min &#34;Off,&#34; and 2 min &#34;On.&#34;</li>
		<li>For primary and secondary antibody incubation steps, run the processor at 3 min &#34;On,&#34; 2 min &#34;Off,&#34; and 3 min &#34;On.&#34; 
		NOTE:&#160;We observe no difference in quality by making the above alterations to the protocol.</li>
	</ol>
	</li>
</ol>
8. Image Analysis
<ol>
	<li>It is recommended to use FIJI &#60;https://fiji.sc/&#62; to conduct image analysis, as it is compatible with multiple file formats. For our data, images in the Nikon ND2 file format were acquired.</li>
	<li>A macro script is provided for easy batch quantification of different parameters pre-selected by FIJI. The following steps are included in the macro: 
	NOTE:&#160;For these examples, &#34;integrated intensity&#34; was measured.</li>
	<li>Open the image files in FIJI and separate channels.</li>
	<li>Z-project each channel stack as a maximum intensity projection.</li>
	<li>Set a lower threshold for each channel. 
	NOTE:&#160;Thresholds should be empirically determined for each experimental data set. While each channel can have a separate lower threshold value, it crucial that channel threshold values are consistently maintained for all images of the same data set.</li>
	<li>Select three to five secondary or tertiary dendrites and save them as regions of interest (ROIs).</li>
	<li>Measure the integrated density of each ROI in surface and intracellular channels.</li>
	<li>Normalize the signal for each ROI by dividing the integrated density value of the surface channel by the intracellular channel.</li>
	<li>Repeat the measurements for all control and experimental images and normalize experimental values to control values (e.g., GluN2B WT or no-glycine conditions).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 mm dia. #1.5 thick coverglasses</td>
			<td>Neuvitro</td>
			<td>GG181.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 555-conjugated goat anti-mouse secondary</td>
			<td>Life Technologies</td>
			<td>A21424</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 555-conjugated goat anti-rabbit secondary</td>
			<td>Life Technologies</td>
			<td>A21429</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 647-conjugated goat anti-mouse secondary</td>
			<td>Life Technologies</td>
			<td>A21236</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa 647-conjugated goat anti-rabbit secondary</td>
			<td>Life Technologies</td>
			<td>A21245</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom Cell Culture Plates</td>
			<td>Corning</td>
			<td>3513</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynasore</td>
			<td>Tocris</td>
			<td>2897</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Tocris</td>
			<td>219</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit Fab fragments</td>
			<td>Sigma</td>
			<td>SAB3700970</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-GluA1 antibody</td>
			<td>Millipore</td>
			<td>MAB2263</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S6546</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal Media</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>NGS</td>
			<td>Abcam</td>
			<td>Ab7481</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM999</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pelco BioWave</td>
			<td>Ted Pella</td>
			<td>36500</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Alfa Aesar</td>
			<td>43368</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Tocris</td>
			<td>1128</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine hydrobromide</td>
			<td>Sigma</td>
			<td>P7280</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Life Technologies</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFP antibody</td>
			<td>Invitrogen</td>
			<td>A11122</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-PSD-95 antibody</td>
			<td>Cell Signaling</td>
			<td>2507</td>
			<td></td>
		</tr>
		<tr>
			<td>Strychnine</td>
			<td>Tocris</td>
			<td>2785</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost plus microscope slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Tocris</td>
			<td>1078</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Chiu, A. M., et al. <a href="https://app.jove.com/v/59982">An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures.</a> J. Vis. Exp. (2019)
This video demonstrates an antibody-feeding approach to study the trafficking of glutamate receptors in primary hippocampal neuronal cultures. This approach allows the observation of receptor populations at both the plasma and internal membranes. Discrimination between these subsets is achieved by labeling the receptors before and after membrane permeabilization, using the same primary antibody but secondary antibodies conjugated to different fluorophores.

<img alt="Figure 1" src="/files/ftp_upload/22129/22129_Figure1.jpg" /> 
Figure 1: cLTP increases surface expression of GluA1. Primary hippocampal neurons at DIV21 were subjected to chemical LTP (cLTP) by incubating with glycine-containing ECS. Distinct labeling of surface-expressed (red) vs. intracellular (blue) GluA1 populations reveals the expected increase in surface expression of AMPAR. (A) Single plane and (B) Z-stacked (maximum intensity projection) confocal pictures. Scale bars = 50 &#181;m (whole cell) or 5 &#181;m (dendrite). Graph shows the increased surface expression of GluA1 after cLTP protocol. Surface expression index: surface/intracellular receptors (n = 3; number of cells: con = 7; cLTP = 7; values represent mean &#177; SEM; ****p &#60; 0.0001 using Mann-Whitney U test). (C) Control experiment in which the permeabilization step was skipped. In addition to surface and internal GluA1, the intracellular excitatory synaptic marker PSD-95 was evaluated. Scale bars = 50 &#181;m (whole cell) or 5 &#181;m (dendrite).
<img alt="Figure 2" src="/files/ftp_upload/22129/22129_Figure2.jpg" /> 
Figure 2: Phosphorylation of GluN2B at S1480 promotes NMDAR internalization. Primary hippocampal neurons were transfected with either GFP-GluN2B WT or the phospho-mimetic mutant GFP-GluN2B S1480E on DIV11-12. Following 3-4 days of protein expression, surface GFP was labeled on live cells with a rabbit anti-GFP antibody and cells were then returned to 37 &#176;C to allow for receptor internalization by endocytosis. Surface-expressed exogenous receptors were visualized with Alexa 555-conjugated secondary antibody, and the internalized population identified after permeabilization using Alexa 647-conjugated antibody. For clarity, surface GFP-GluN2B is pseudocolored in green and internalized GFP-GluN2B is pseudocolored in white. (A) Single plane and (B) Z-stacked (maximum intensity projection) confocal pictures. Scale bars = 50 &#181;m (whole cell) or 5 &#181;m (dendrite). Graph shows the elevated internalization displayed by the phospho-mimetic mutant GluN2B S1480E. Internalization index: internalized receptors/surface-expressed receptors (n = 6; number of cells: WT = 34; S1480E = 28; values represent mean &#177; SEM; ***p &#60; 0.001 using Mann-Whitney U test). (C) Control experiment in which the internalization step (Intenaliz.) was performed at 4 &#176;C. Scale bars = 50 &#181;m (whole cell) or 5 &#181;m (dendrite).
<img alt="Figure 3" src="/files/ftp_upload/22129/22129_Figure3.jpg" /> 
Figure 3: Phosphorylation of GluN2B at S1480 does not modify NMDAR recycling. Primary hippocampal neurons were transfected with either GFP-GluN2B WT or the phospho-mimetic mutant GFP-GluN2B S1480E on DIV11-12 as shown in Figure 3. Following 3-4 days of protein expression, surface GFP was labeled on live cells with a rabbit anti-GFP antibody and cells were then returned to 37 &#176;C to allow for receptor internalization by endocytosis. Remaining surface-expressed receptors were blocked by Fab incubation and recycling was allowed for 45 min. Available surface expressed exogenous receptors (recycled) were visualized with Alexa 555-conjugated secondary antibody, and the internalized population identified after permeabilization using Alexa 647-conjugated antibody. For clarity, surface GFP-GluN2B is pseudocolored in white and internalized GFP-GluN2B is pseudocolored in green. (A) Single plane and (B) Z-stacked (maximum intensity projection) confocal pictures. Scale bars = 50 &#181;m (whole cell) or 5 &#181;m (dendrite). Graph shows the lack of effect the GluN2B S1480 phosphorylation has on recycling. Recycling index: recycled receptors/internalized receptors (n = 5; number of cells: WT = 27; S1480E = 24; values represent mean &#177; SEM; n.s. = non-significant using Mann-Whitney U test). (C) Control experiment in which the Fab incubation step to block surface-expressed epitopes was skipped. Scale bars = 50 &#181;m (whole cell) or 5 &#181;m (dendrite).

1. Preparation Before Labeling

	Preparation and maintenance of primary hippocampal cultures&#8203;
	
		Prepare primary hippocampal cultures at a density ...

Take three samples of hippocampal neuronal cultures on coverslips.
Introduce primary antibodies to label the surface-expressed glutamate receptors.
Add a conditioned medium to the second and third samples to induce endocytosis of the antibody-bound receptors.
On the third sample, add Fab fragments to block epitopes on the non-endocytosed antibodies.
Introduce an inhibitor to block receptor endocytosis while allowing receptor recycling to the surface.
In all samples, fix the cells and add a blocking solution to prevent non-specific labeling.&#38;
Introduce fluorophore-tagged secondary antibodies to label the primary antibodies on the surface-bound receptors, including the recycled ones.
Permeabilize the cellular membranes.
Add the primary antibody again to the first sample to label the pre-existing endocytosed receptors.
Introduce a different fluorophore-tagged secondary antibody to all samples to label the endocytosed receptor-bound primary antibodies, distinguishing between endocytosed and surface-bound receptors.
Using a confocal microscope, quantify the surface-expressed, endocytosed, and recycled receptors in the different treatments.

34 조회수. 해마 뉴런에서 글루타메이트 수용체 이동을 연구하기 위한 체외 기술

비디오: 해마 뉴런에서 글루타메이트 수용체 이동을 연구하기 위한 체외 기술 - 실험

Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as valency, spatial arrangement, interdomain distance, and Fc mutations affect the anti-tumor efficacy of these therapies, commonly by influencing the homing of T cells to tumors, which remains a major challenge. Here, we describe a method to transduce activated human T cells with luciferase, allowing in vivo tracking of T cells during T-BsAb therapy studies. The ability of T-BsAbs to redirect T cells to tumors can be quantitatively evaluated at multiple time points during treatment, allowing researchers to correlate the anti-tumor efficacy of T-BsAbs and other interventions with the persistence of T cells in tumors. This method alleviates the need to sacrifice animals during treatment to histologically assess T cell infiltration and can be repeated at multiple time points to determine the kinetics of T cell trafficking during and after treatment.

Here, we describe a method for transducing human T cells with luciferase to facilitate in vivo tracking of bispecific antibody-induced T cell trafficking to tumors in studies to evaluate the anti-tumor efficacy and mechanism of T cell-engaging bispecific antibodies.

루시페라아제 형질도입된 인간 T 세포를 이용한 이중특이적 항체 유도 T 세포 트래피킹 추적

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as ...

연구

JoVE Journal

암 연구학

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (PET/CT).

에 의해 쥐 림프구 라벨링<sup&gt; (64)</sup&gt; CU-항체 수용체는 대한 타겟팅<em&gt; 생체</em&gt; PET / CT에 의한 세포 트래 피킹

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

면역학 및 감염병학

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

AMPA 모니터링을 위한 하이 콘텐츠 분석 결과 수용 체 매매

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons

내레이션 대본

An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons