We thank the UBC Life Sciences Institute (LSI) Imaging Facility for supporting and maintaining the STED microscope. This work was funded by grant #68865 from the Canadian Institutes of Health Research (to M.R.G.). We thank Dr. Kozo Kaibuchi (Nagoya University, Nagoya, Japan) for the CLIP-170-GFP plasmid.

All animal procedures were approved by the University of British Columbia Animal Care Committee.
1. Expressing GFP-fusion proteins in A20 B-lymphoma cells
<ol>
	<li>Culture A20 cells in complete RPMI medium (RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 &#181;M 2-mercaptoethanol, 2 mM glutamine, 1 mM pyruvate, 50 U/mL penicillin, and 50 &#181;g/mL streptomycin) at 37 &#176;C in a tissue culture incubator with 5% CO2.</li>
	<li>Prepare the transfection reagent (see the Table of Materials) according to the manufacturer&#39;s instructions. 
	NOTE: This protocol has been optimized for the specific reagents and transfection protocol described in the Table of Materials. Other transfection reagents that we have tried have not yielded sufficient transfection efficiency.</li>
	<li>Count the cells using a hemocytometer. Centrifuge 2.5 x 106 A20 cells (for each transfection) for 5 min at 525 x g. Resuspend the cells in 100 &#181;L of transfection reagent containing 1-2.5 &#181;g of plasmid DNA. For DNA encoding CLIP-170-GFP23, use 2.5 &#181;g per transfection. Mix gently.</li>
	<li>Transfer the cells to a well of a 6-well plate, and adjust the volume to 2 mL with pre-warmed complete RPMI medium. Culture the cells for 18 h at 37 &#176;C to allow protein expression.</li>
</ol>
2. Isolating Primary Mouse B Cells and Activating Them with LPS
<ol>
	<li>Use sterilized surgical tools to remove the spleen from a mouse, following protocols approved by the institution&#39;s Animal Care Committee.</li>
	<li>In the tissue culture hood, place a sterile 70-&#181;m cell strainer into a 35-mm tissue culture dish containing 3 mL of room temperature sterile PBS. Use the rubber part of the plunger from a 5-ml syringe to crush the spleen through the cell strainer.</li>
	<li>Pipet the single cell suspension of splenoctyes into a sterile 15-mL tube and centrifuge at 525 x g for 5 min.</li>
	<li>Use a magnetic bead-based B cell isolation kit (see Table of Materials) to obtain a highly enriched population of B cells.</li>
	<li>Resuspend the B cells to 3 x 106/mL in complete RPMI medium supplemented with 2.5 &#181;g/mL LPS plus 5 ng/mL B cell activating factor (BAFF; a survival cytokine).</li>
	<li>Culture the cells for 6 hr at 37 &#176;C.</li>
</ol>
3. Coating Glass Coverslips with Anti-Ig Antibodies
<ol>
	<li>Prepare the antibody solution for coating the coverslips. Dilute the stock solution of goat-anti-mouse Ig antibodies into room temperature sterile PBS to make a 12.5 &#181;g/mL solution; 400 &#181;L of antibody solution is required for each coverslip. 
	NOTE: Use goat anti-mouse IgG for A20 cells; use goat anti-mouse IgM for primary B cells. Goat IgG antibodies do not bind well to mouse Fc receptors. However, to avoid any potential Fc receptor binding, one can use F(ab) or F(ab&#39;)2 fragments of anti-mouse IgG or anti-mouse IgM antibodies.</li>
	<li>Dip a #1.5 18-mm round glass coverslip in 100% methanol and let it dry completely (~10 min).</li>
	<li>Using forceps, place the dried coverslip into a 12-well tissue culture plate and pipet 400 &#181;L of the 12.5 &#181;g/mL antibody solution (5 &#181;g total; 2 &#181;g/cm2) onto the coverslip so that it forms a bubble at the center of the coverslip and spreads to the edges.</li>
	<li>Be careful to cover the entire coverslip, but do not allow the antibody solution to extend past the edge of the glass coverslip and onto the tissue culture plastic. Incubate at room temperature for 30 min.</li>
	<li>Wash the coverslips by pipetting 1 mL of sterile PBS into the well and subsequently aspirating out the solution. Repeat two more times to remove unbound antibodies.</li>
	<li>Add 1 mL of sterile PBS to the well containing the antibody-coated coverslip until cells are ready to be added. 
	NOTE: The coverslips can be kept at room temperature, covered with PBS, for use on the same day.</li>
</ol>
4. B Cell Spreading on Anti-Ig-Coated Coverslips
<ol>
	<li>Prepare modified HEPES-buffered saline (mHBS): 25&#8197;mM HEPES, pH 7.2, 125&#8197;mM NaCl, 5&#8197;mM KCl, 1&#8197;mM CaCl2, 1&#8197;mM Na2HPO4, 0.5&#8197;mM MgSO4, 1&#8197;mg/mL dextrose, 2&#8197;mM glutamine, 1&#8197;mM sodium pyruvate, and 50&#8197;&#181;M 2-mercaptoethanol). Filter sterilize this solution and store at 4 &#176;C.</li>
	<li>On the day of the experiment, make 50 mL of mHBS with 2% FBS (mHBS-FBS) by adding 1 mL of heat-inactivated FBS to 50 mL mHBS. Warm the mHBS-FBS to 37 &#176;C before use.</li>
	<li>Centrifuge the transfected A20 cells or the primary B cells that had been cultured with BAFF plus LPS at 525 x g for 5 min.</li>
	<li>Resuspend the cells in 1 mL of mHBS-FBS, count the cells using a hemocytometer, and dilute the cells to 2 x 105 cells/mL in mHBS-FBS.</li>
	<li>Using forceps, transfer the coverslip from the tissue culture plate to a piece of paraffin film.</li>
	<li>Add 250 &#181;L of B cells (5 x 104 cells) to each coverslip.</li>
	<li>Incubate the coverslips at 37 &#176;C for 15 min in the dark to allow the B cells to spread over the anti-IgG-coated surface.</li>
</ol>
5. Fixing and Immunostaining the Cells
<ol>
	<li>Prepare the fixation solution by diluting the 16% paraformaldehyde stock solution and the 50% glutaraldehyde stock solution in room-temperature distilled water to yield final concentrations of 3% paraformaldehyde and 0.1% glutaraldehyde. Prepare the fixation solution on the day it is to be used and discard any excess. 
	CAUTION: The paraformaldehyde and glutaraldehyde stock solutions should only be used in a chemical fume hood. Follow the instructions on the material safety data sheet (MSDS).</li>
	<li>Prepare the permeabilization/blocking buffer (3% BSA and 0.1% Triton X-100 diluted in PBS). Filter sterilize this solution and store at 4 &#176;C. On the day of the experiment, warm the required amount to room temperature.</li>
	<li>Prepare the staining buffer (1% BSA and 0.1% saponin in PBS). Sterilize this solution using a 0.2 &#181;m filter and store at 4 &#176;C. On the day of the experiment, warm the required amount to room temperature.</li>
	<li>Pipet 350 &#181;L of the fixation solution onto each coverslip, ensuring that the surface is completely covered. Incubate at room temperature for 10 min in the dark.</li>
	<li>Remove the fixation solution from the coverslip by carefully aspirating from the edge of the coverslip using a micropipet. 
	CAUTION: The fixation solution should be discarded as hazardous chemical waste.</li>
	<li>Wash the coverslips once with 500 &#181;L of permeabilization/blocking buffer. Pipet off the liquid.</li>
	<li>Permeabilize and block the cells by adding 250 &#181;L of permeabilization/blocking buffer to each coverslip, and incubating for 10 min at room temperature in the dark.</li>
	<li>Dilute the rabbit anti-tubulin antibody 1:100 in staining buffer.</li>
	<li>Aspirate off the permeabilization/blocking buffer from the coverslips, and add 50 &#181;L of the diluted anti-tubulin antibody solution to each coverslip. Incubate at room temperature for 30 min in the dark.</li>
	<li>Prepare the secondary antibody/actin stain solution by diluting both the Alexa Fluor 532-conjugated goat anti-rabbit IgG secondary antibody and Alexa Fluor 568-conjugated phalloidin 1:100 in staining buffer.</li>
	<li>Wash the coverslips 3 times to remove excess primary antibody by adding 500 &#181;L of staining buffer and then aspirating it.</li>
	<li>Add 50 &#181;L of the secondary antibody/actin stain solution to each coverslip, and incubate for 30 min at room temperature in the dark.</li>
	<li>Wash the coverslips 3 times to remove excess secondary antibody by adding 500 &#181;L of staining buffer and then aspirating it.</li>
	<li>Add 10 &#181;L of mounting reagent to a microscope slide. Mount the coverslip onto the microscope slide with the cell side down. 
	NOTE: An &#34;anti-fade&#34; mounting medium (see Table of Materials) is strongly recommended to preserve the fluorescence intensity of GFP fusion proteins. Avoid the use of mounting reagents containing DAPI, which can interfere with the imaging of the fluorophores when using the STED laser.</li>
	<li>Allow the slides to dry overnight in the dark. Image the slides the next day. 
	NOTE: Imaging the slides as soon as the coverslip is dry is strongly recommended, as the GFP fluorescence will decrease over time.</li>
</ol>
6. Imaging Using the STED Microscope
NOTE: Please note that all software steps described below are specific to the microscope and software we used (see the Table of Materials). The steps and settings will need to be adjusted if imaging is performed using a different microscope/software.
<ol>
	<li>Turn on the STED microscope, activate the lasers and epifluorescence illumination lamp, and start up the microscope software.</li>
	<li>Set the parameters for the STED depletion lasers. 
	NOTE: This will depend on the specific microscope being used and the lasers that it is equipped with. Some recommended settings are white light (WLL) 70%; 592 nm laser 80%; 660 nm laser 80%.</li>
	<li>Activate the STED settings and align the STED laser beams using the 100X objective.</li>
	<li>Using the eyepiece, focus the sample and select a cell with moderate GFP expression. 
	NOTE: Low GFP expression will not be visible because STED reduces the emission intensity. Conversely, high expression of CLIP-170-GFP induces the spontaneous formation of large clusters, which does not reflect the normal microtubule plus-end protein complex morphology and distribution. Selecting a cell with moderate GFP expression is essential for accurately imaging the cytoskeletal structures at the antigen contact site.</li>
	<li>Zoom into the selected cell and choose the region of interest to be imaged. Adjust the focus so that the x-y plane of the B cell that is closest to the coverslip is in focus.
	<ol>
		<li>To do this, move the objective progressively closer to the coverslip so that the B cell cytoskeletal structures come into focus and then go out of focus. Then gradually move the objective in the opposite direction until the B cell cytoskeletal structures first come back into focus.</li>
	</ol>
	</li>
	<li>Optimize the settings including the laser power, excitation beam, and detector range. Start with the settings recommended by the manufacturer&#39;s instructions. Then make adjustments as needed to optimize the signal for the samples. Image all samples to be compared to each other with the same settings.</li>
	<li>Under the &#34;acquire&#34; tab of the software, set the image acquisition to sequential frame acquisition in the lower left &#34;Sequential Scan&#34; panel by checking the option &#34;between frames&#34;.</li>
	<li>Set the acquisition sequence. 
	Note: We acquire the GFP fluorescence first in order to avoid degradation of the GFP signal. 
	Depending on the combination of fluorophores that are used in addition to GFP, imaging the longer wavelength fluorescence signal first may yield better results. However, the order in which the fluorophores or fluorescent proteins are subjected to stimulated emission depletion and imaged should be optimized to obtain the strongest fluorescence signals and best resolution.</li>
	<li>Select and set the STED laser power for each fluorophore under the &#34;Acquire&#34; tab of the software, and use the slider bar for either the 592-nm or the 660-nm STED laser to adjust the laser power. Adjust the power for the 592-nm depletion laser for GFP and for Alexa Fluor 532. Adjust the power for the 660-nm depletion laser for Alexa Fluor 568.</li>
	<li>To increase the resolution and reduce the background signal, go to the &#34;Acquisition&#34; settings under the &#34;Acquire&#34; tab of the software, increase the line and/or frame averaging by selecting a value greater than 1 using the drop-down menu for each option.</li>
	<li>Also, acquire the fluorescent signal using time-gated STED by checking the gating option for the fluorophore under the &#34;Acquire&#34; tab, and specifying values for time-gating (see note below). Increase the STED laser power to enhance the resolution, though this will also increase photobleaching of the fluorophores. 
	NOTE: The gating time will need to be optimized for the microscope, sample, and fluorophores being used. A time gating of 0.3 to 6 ns is recommended. After fixation, GFP is particularly sensitive to high laser power. To reduce the photobleaching of GFP, time gating should be applied and the STED laser power should be reduced.</li>
	<li>To capture 3D STED images, use the slider to change the point spread function (PSF) to &#34;3D STED&#34;.</li>
	<li>Manually acquire multiple images to ensure reproducibility. Deconvolve the images (see Figure 1) using a deconvolution software package41(see Table of Materials).</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Rap+GTPases+in+B+lymphocytes+modulates+B+cell+antigen+receptor-induced+activation+of+Akt+but+has+no+effect+on+MAPK+activation.">Activation of the Rap GTPases in B lymphocytes modulates B cell antigen receptor-induced activation of Akt but has no effect on MAPK activation.</a> J Biol Chem. 278 (43), 41756-41767 (2003).</li><li>Batista, F. D., Iber, D., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cells+acquire+antigen+from+target+cells+after+synapse+formation.">B cells acquire antigen from target cells after synapse formation.</a> Nature. 411 (6836), 489-494 (2001).</li><li>Treanor, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+cortical+actin+remodeling+by+ERM+proteins+controls+BCR+microcluster+organization+and+integrity.">Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity.</a> J Exp Med. 208 (5), 1055-1068 (2011).</li><li>Mattila, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+and+tetraspanin+networks+organize+receptor+nanoclusters+to+regulate+B+cell+receptor-mediated+signaling.">The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling.</a> Immunity. 38 (3), 461-474 (2013).</li><li>Yuseff, M. -. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+Secretion+of+Lysosomes+at+the+B+Cell+Synapse+Couples+Antigen+Extraction+to+Processing+and+Presentation.">Polarized Secretion of Lysosomes at the B Cell Synapse Couples Antigen Extraction to Processing and Presentation.</a> Immunity. 35 (3), 361-374 (2011).</li><li>Mace, E. M., Orange, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+immunological+synapse+by+dual+color+time-gated+stimulated+emission+depletion+(STED)+nanoscopy.">Visualization of the immunological synapse by dual color time-gated stimulated emission depletion (STED) nanoscopy.</a> J Vis Exp. (85), (2014).</li><li>Russ, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Image Processing Handbook, Sixth Edition. , (2011).</li><li>Stinchcombe, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+polarization+delivers+secretory+granules+to+the+immunological+synapse.">Centrosome polarization delivers secretory granules to the immunological synapse.</a> Nature. 443 (7110), 462-465 (2006).</li><li>Vicidomini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sharper+low-power+STED+nanoscopy+by+time+gating.">Sharper low-power STED nanoscopy by time gating.</a> Nat Methods. 8 (7), 571-573 (2011).</li></ol>

The authors have nothing to disclose.

Detailed images of cytoskeletal structures can be obtained using STED super-resolution microscopy, which can theoretically achieve a resolution of 50 nm, compared to conventional confocal microscopy, for which the diffraction-limited resolution is ~200 nm24. The ability to resolve finer structures is further enhanced by using deconvolution software to calculate the most likely position of the original light source from the observed &#34;blurred&#34; fluorescence signal. This protocol describes met...

When B cells bind to polarized arrays of antigens (e.g., displayed on the surface of antigen-presenting cells (APCs)), the resulting B-cell receptor (BCR) signaling drives the formation of a classic immune synapse structure, which was first described in T cells1,2,3,4,5,6,7,8,9,10,11,12,13. Initially, microclusters of antigen-bound BCRs form at the periphery of the B cell:APC contact site. These microclusters then move towards the center of the antigen contact site, where they coalesce into a central supramolecular activation cluster (cSMAC) that forms the core of the immune synapse. Immune synapse formation optimizes BCR signaling and facilitates BCR-mediated antigen extraction from the APC membrane14. This antigen acquisition, which is followed by BCR-mediated antigen internalization and subsequent antigen processing, allows B cells to present peptide:MHC II complexes to T cells and elicit T cell help14. Because immune synapse formation promotes B cell activation, elucidating the mechanisms that establish this functional pattern of receptor organization can provide new insights into how humoral immune responses are initiated and regulated.
Reorganization of both the actin and microtubule cytoskeletons is essential for immune synapse formation. Localized BCR signaling stimulated by a spatially-polarized array of antigens induces rapid and dramatic remodeling of the actin cytoskeleton1,15. The formation of dendritic actin structures at the periphery of the B cell exerts pushing forces on the plasma membrane and promotes B cell spreading. This allows the B cell to scan a greater area of the antigen-bearing surface, and increases the number of BCRs that bind antigen and activate BCR signaling pathways. At the same time, the MTOC and the microtubule network are reoriented towards the site of antigen contact. As the MTOC approaches the antigen contact site, microtubules emanating from the MTOC extend along the inner face of the plasma membrane at the interface between the B cell and the antigen-bearing surface16,17. These juxtamembrane microtubules can then act as tracks for the dynein-mediated centripetal movement of antigen-bound BCR microclusters18, leading to the formation of a cSMAC.
The reorientation and polarization of the MTOC towards the immune synapse requires intact actin and microtubule cytoskeletons, and often depends on interactions between the cortical actin network and microtubules16,17,19,20. Cortical actin-binding proteins, such as IQGAP1, can capture microtubules by interacting with protein complexes that decorate the microtubule plus-ends21. These dynamic complexes of plus-end binding proteins include EB1 and CLIP-170, which are collectively referred to as microtubule plus-end tracking proteins (+TIPs)21,22. +TIPs at the ends of microtubules can bind to proteins that are associated with either the plasma membrane or the cortical actin cytoskeleton. This allows force-generating mechanisms (e.g., the minus-end directed movement of cortically-anchored dynein along microtubules) to exert pulling forces on microtubules, and thereby reposition the MTOC. CLIP-170 can bind to the actin-associated scaffolding protein IQGAP123, and we have shown that both of these proteins are required for BCR-induced MTOC polarization towards the immune synapse17. This IQGAP1-CLIP-170 interaction may play a key role in coordinating the remodeling of the actin cytoskeleton with the repositioning of the microtubule network at the B-cell immune synapse.
Conventional fluorescence microscopy has revealed the dramatic reorganization of the actin and microtubule cytoskeletons during B-cell immune synapse formation2. However, this approach cannot resolve small cellular structures in detail due to the diffraction limit of light, which, according to Abbe&#39;s law, is dependent on the wavelength of light used to illuminate the sample and the aperture of the objective24. This diffraction limit constrains the resolution of conventional light microscopes to 200-300 nm in the lateral direction and 500-700 nm in the axial direction25. Therefore, smaller subcellular structures, as well as the fine details of the actin and microtubule cytoskeletons, could only be observed using electron microscopy. Electron microscopy imaging of the cytoskeleton is time consuming, requires harsh sample fixation and preparation protocols that can alter biological structures, and is limited to antibody-mediated detection. The ability to immunostain and simultaneously image multiple proteins or cellular structures is a substantial advantage of fluorescence microscopy. Moreover, expressing fluorescent fusion proteins in cells enables real-time imaging and is useful when effective antibodies for immunostaining the protein of interest are not available.
Recent technological advances in super-resolution microscopy have overcome the diffraction limits of light and allowed the visualization of nanoscale cellular structures24. One such super-resolution microscopy technique is called stimulated emission depletion (STED) microscopy. STED employs two lasers, where one laser excites the fluorophore and a second laser with a donut-shaped pattern selectively suppresses the fluorescence emission around the fluorophore. This reduces the point-spread function (apparent area) of a single fluorescent particle and provides a sub-diffraction limit fluorescent image25,26. Ground-state depletion microscopy also employs fluorescence-based techniques to acquire super-resolution images. However, the image acquisition and reconstruction times are long, there are only a limited number of fluorophores that can be used, and the simultaneous high-resolution imaging of multiple cytoskeletal components is technically challenging because maintaining actin and microtubule structures requires different fixation procedures. Therefore, STED has multiple advantages over electron microscopy and other super-resolution microscopy approaches in that it offers rapid image acquisition, has minimal post-processing requirements, and employs the same fluorophores and staining techniques that are used for conventional fluorescence microscopy of fixed samples26.
Super-resolution microscopy has now been used to visualize actin structures at the immune synapse in natural killer (NK) cells and T cells26,27,28,29,30,31. However, super-resolution imaging of the microtubule cytoskeleton, as well as the coordinated reorganization of the actin and microtubule cytoskeletons during immune synapse formation, has only recently been reported17. We used STED microscopy to image B cells that had been allowed to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which stimulate BCR signaling and initiate cytoskeleton reorganization. When plated on immobilized anti-Ig antibodies, B cells undergo dramatic actin-dependent spreading, which recapitulates the initial events during immune synapse formation. Importantly, STED microscopy revealed the fine details of the dendritic ring of F-actin that forms at the periphery of the immune synapse and showed that the MTOC, as well as the microtubules attached to it, had moved close to the antigen contact site17. These microtubules extended outward towards the peripheral ring of F-actin. Moreover, multi-color STED imaging of various combinations of F-actin, tubulin, IQGAP1, and GFP-tagged CLIP-170 +TIPs showed that microtubule plus-ends marked by CLIP-170-GFP were closely associated with the peripheral actin meshwork and with IQGAP1, a cortical capture protein17.
Here, we present a detailed protocol for imaging the actin and microtubule cytoskeletons at the immune synapse using STED microscopy. These methods have been optimized using the A20 murine B cell line, which has been widely employed for studying BCR signaling and immune synapse formation17,32,33,34,35,36,37,38,39. Because commercial antibodies to CLIP-170 did not work well for immunostaining in previous experiments, we describe in detail the expression of GFP-tagged CLIP-170 in A20 cells, along with staining protocols for simultaneously visualizing up to three cytoskeletal components or cytoskeleton-associated proteins. Methods for using STED microscopy to image actin at NK cell immune synapses have been described previously40. Here, we extend this to acquiring multi-color super-resolution images of both the actin and microtubule cytoskeletons in B cells.
A critical consideration for super-resolution microscopy is using the appropriate fixation procedures for maintaining cellular structures and preventing damage to fluorescent proteins. The fixation and staining methods presented herein have been optimized to retain GFP fluorescence and provide high-resolution imaging of the actin and microtubule networks. When expressing fluorescent proteins, it should be noted that B cells are usually difficult to transfect. Using this protocol, 20-50% of A20 cells typically express the transfected GFP fusion protein, and among this population the levels of protein expression are variable. Nevertheless, super-resolution imaging of actin and microtubules using the procedures we describe is quite robust and high-quality images are readily obtained. Despite their small size relative to A20 cells, we show that these procedures can also be used to image the microtubule network in primary B cells that have been briefly activated with lipopolysaccharide (LPS). We have shown that LPS-activated primary B cells can be transfected with siRNAs at relatively high efficiency (i.e., such that protein depletion can be detected by immunoblotting), making them a good alternative to the use of B cell lines for some studies17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A20 mouse B-lymphoma cells</td>
			<td>ATCC</td>
			<td>TIB-208</td>
			<td>Murine B-cell lymphoma of Balb/c origin that expresses an IgG-containing BCR on its surface</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Thermo Fisher Scientific</td>
			<td>&#160;R0883</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td>Heat inactivate at 56 oC for 30 min</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Millipore Sigma</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Millipore Sigma</td>
			<td>G5763</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Millipore Sigma</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td>Liquid, 10,000 units</td>
		</tr>
		<tr>
			<td>Additional materials for primary B cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#181;m cell strainer</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic bead-based B cell isolation kit</td>
			<td>Stemcell Technologies</td>
			<td>19854</td>
			<td>EasySep Mouse B cell Isolation kit</td>
		</tr>
		<tr>
			<td>Lipopolysaccharide (LPS)</td>
			<td>Millipore Sigma</td>
			<td>L4391</td>
			<td>LPS from E. coli 0111:B4</td>
		</tr>
		<tr>
			<td>B cell-activating factor (BAFF)</td>
			<td>R&#38;D Systems</td>
			<td>2106-BF-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid encoding CLIP-170-GFP</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Kozo Kaibuchi (Nagoya Univ., Nagoya, Japan); described in Fukata et al. Cell 109:873-885, 2002</td>
		</tr>
		<tr>
			<td>Recommended transfection reagents and equipment </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amaxa Nucleofection kit V</td>
			<td>Lonza</td>
			<td>VCA-1003</td>
			<td>Follow the manufacturer&#39;s directions for mixing the transfection reagents with the DNA</td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector model 2b</td>
			<td>Lonza</td>
			<td>AAB-1001</td>
			<td>Program L-013 used</td>
		</tr>
		<tr>
			<td>Falcon 6-well plates, TC treated, sterile</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Coating coverslips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18-mm diameter round #1.5 cover glasses</td>
			<td>Thomas Scientific</td>
			<td>1217N81</td>
			<td>Similar product: Marienfield-Superior catalogue #117580</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>1381242</td>
			<td>Dissecting extra fine splinter forceps</td>
		</tr>
		<tr>
			<td>Falcon 12-well sterile tissue polystyrene tissue culture plate</td>
			<td>Corning</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>100% methanol</td>
			<td>Fisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile phosphate buffered saline (PBS) without calcium or magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti-mouse IgG antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-008</td>
			<td>For A20 cells</td>
		</tr>
		<tr>
			<td>Goat-anti-mouse IgM antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-005-020</td>
			<td>For primary mouse B cells</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Staining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (16% stock solution)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Dilute with PBS to working concentration</td>
		</tr>
		<tr>
			<td>Glutaraldehyde (50% stock solution)</td>
			<td>Millipore Sigma</td>
			<td>340855</td>
			<td>Dilute with PBS to working concentration</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Millipore Sigma</td>
			<td>S2149</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA), Fraction V</td>
			<td>Millipore Sigma</td>
			<td>10735094001</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-tubulin antibody</td>
			<td>Abcam</td>
			<td>ab52866</td>
			<td>1:250 dilution recommended but should be optimized</td>
		</tr>
		<tr>
			<td>Alexa Fluor 532-conjugated goat anti-rabbit IgG</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11009</td>
			<td>1:100 dilution recommended but should be optimized</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568-conjugated phalloidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A12380</td>
			<td>1:100 dilution recommneded but should be optimized</td>
		</tr>
		<tr>
			<td>Prolong Diamond Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36970</td>
			<td>Without DAPI</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>VWR</td>
			<td>P1150-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Millipore Sigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Millipore Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Millipore Sigma</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M63</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 TCS STED microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Huygens deconvolution software</td>
			<td>Scientific Voume Imaging</td>
			<td></td>
			<td>See https://svi.nl/HuygensProducts</td>
		</tr>
	</tbody>
</table>

visualizing the actin and microtubule cytoskeletons at the b-cell immune synapse using stimulated emission depletion (sted) microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure that optimizes B-cell receptor (BCR) signaling and BCR-mediated antigen acquisition. Both the remodeling of the actin cytoskeleton and the reorientation of the microtubule network towards the antigen contact site are essential for immune synapse formation. Remodeling of the actin cytoskeleton into a dense peripheral ring of F-actin is accompanied by polarization of the microtubule-organizing center towards the immune synapse. Microtubule plus-end binding proteins, as well as cortical plus-end capture proteins mediate physical interactions between the actin and microtubule cytoskeletons, which allow them to be reorganized in a coordinated manner. Elucidating the mechanisms that control this cytoskeletal reorganization, as well as understanding how these cytoskeletal structures shape immune synapse formation and BCR signaling, can provide new insights into B cell activation. This has been aided by the development of super-resolution microscopy approaches that reveal new details of cytoskeletal network organization. We describe here a method for using stimulated emission depletion (STED) microscopy to simultaneously image actin structures, microtubules, and transfected GFP-tagged microtubule plus-end binding proteins in B cells. To model the early events in immune synapse formation, we allow B cells to spread on coverslips coated with anti-immunoglobulin (anti-Ig) antibodies, which initiate BCR signaling and cytoskeleton remodeling. We provide step-by-step protocols for expressing GFP fusion proteins in A20 B-lymphoma cells, for anti-Ig-induced cell spreading, and for subsequent cell fixation, Immunostaining, image acquisition, and image deconvolution steps. The high-resolution images obtained using these procedures allow one to simultaneously visualize actin structures, microtubules, and the microtubule plus-end binding proteins that may link these two cytoskeletal networks.

For B cells spreading on immobilized anti-Ig, STED microscopy used in conjunction with deconvolution software provides higher resolution images of cytoskeletal structures than confocal microscopy. This is evident in Figure 1, where the F-actin network was visualized using the protocol described above. A comparison of confocal and STED super-resolution images of the same sample shows that the STED images are of higher resolution and reveal more detailed struct...

Watch this Scientific Journal Video about Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy at JoVE.com

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion (STED) Microscopy

B cells that bind to membrane-bound antigens (e.g., on the surface of an antigen-presenting cell) form an immune synapse, a specialized cellular structure ...

visualizing-actin-microtubule-cytoskeletons-at-b-cell-immune-synapse

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JoVE Journal

Immunology and Infection

13.6K Views.  University of British Columbia. We present a protocol for using STED microscopy to simultaneously image actin structures, microtubules, and microtubule plus-end binding proteins in B cells that have spread on coverslips coated with antibodies to the B-cell receptor, a model for the initial phase of immune synapse formation.

Video: Visualizing the Actin and Microtubule Cytoskeletons at the B-cell Immune Synapse Using Stimulated Emission Depletion STED Microscopy

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.