Name: Video: Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy
Uploaded: 2014-03-24T20:00:00.000+00:00
Duration: 10 min

We thank Geoff Daniels for technical assistance. This work was funded by R01 AI067946 to J.S.O.

1. Coat Coverslips with Antibody
<ol>
	<li>Prewarm (at 37 &#176;C) 30 ml of RPMI 10% FCS media and 1 ml of BD Cytofix/Cytoperm.</li>
	<li>Prepare a solution of 5 &#956;g/ml of purified antibody in phosphate buffered saline (PBS). For activation of the NK92 cell line, use of anti-CD18&#160; and anti-NKp30 is recommended.
	<ol>
		<li>Mark one approximately dime sized circle for each condition on a #1.5 coverslip using a PAP pen. For a dual color experiment, there should be four conditions: unstained, dual stained, and two single stained conditions. Dispense 200 &#181;l of antibody solution in each region and incubate at 37 &#176;C for 30 min.</li>
		<li>Wash coverslips by gently immersing each one in 50 ml of PBS in a 50 ml conical tube at room temperature. Washing should occur immediately prior to the addition of cells and care should be taken to avoid antibody drying on the coverslip.</li>
	</ol>
	</li>
</ol>
2. Activate NK Cells on Coverslips; Fix and Permeabilize
<ol>
	<li>Isolate 5 x 105 NK92 cells per condition. Centrifuge and decant supernatant. Wash once with 10 ml prewarmed media from step 1.1. Centrifuge and decant supernatant.
	<ol>
		<li>Resuspend cells in prewarmed media from step 1.1 at a concentration of 2.5 x 106/ml.</li>
		<li>Gently decant 200 &#181;l to the center of the region created in section&#160;1.2.1. Incubate at 37 &#176;C for 20 min at 5% CO2. (Note: this time can be extended or decreased depending on the biological function of interest. For NK cell granule polarization, 20 min is sufficient).</li>
	</ol>
	</li>
	<li>Following incubation of cells, gently wash coverslips by immersing each in 50 ml of room temperature PBS in a 50 ml conical tube.</li>
	<li>Add 1 &#181;l of Triton X-100 to 1 ml of prewarmed Fix/Perm solution from step 1.1 and vortex thoroughly. Fix and permeabilize by adding 200 &#181;l of Fix/Perm buffer (step 2.3) to cells. Incubate for 10 min in the dark at room temperature.</li>
</ol>
3. Stain Cells
<ol>
	<li>Prepare staining buffer: Phosphate buffered saline (PBS), 1% BSA, 0.1% Saponin.
	<ol>
		<li>Prepare solution of primary antibody in 200 &#181;l staining buffer (see step&#160;3.1). (Note: antibody should be titrated prior to use). Avoid the use of primary antibody that is raised in the same species used to coat the coverslip (step 1.2). Also avoid Strepatividin-biotin linkages for STED imaging.</li>
		<li>Following section 2.3.1, gently wash coverslips in 50 ml staining buffer. Dab edges of PAP-pen region with cotton swab to remove excess buffer. Apply antibody solution created in section 3.1.1. Incubate 30 min in the dark at room temperature. (Recommended: incubate coverslips in slide box with a moist paper towel to maintain humidity).</li>
	</ol>
	</li>
	<li>Prepare solution of secondary antibody in 200 &#181;l staining buffer. Recommended fluorophores are Alexa Fluor 488, Pacific Orange, and V500. Generally, a 1:200 dilution is suitable for STED imaging.
	<ol>
		<li>Gently wash coverslips in 50 ml staining buffer. Dab edges of PAP-pen region with cotton swab to remove excess buffer. Apply secondary antibody solution. Incubate 30 min in the dark at room temperature.</li>
	</ol>
	</li>
	<li>Repeat washing and staining for additional proteins of interest. If detecting F-actin with Phalloidin, this can be included with secondary antibody, generally at a 1:200 dilution.</li>
</ol>
4. Mount Coverslips on Slides
<ol>
	<li>Prepare mounting medium. Note: Prolong or Prolong Gold are preferable. VECTASHIELD must be avoided, as it is not compatible with STED. 2,2-thiodioethanol must be avoided if Phalloidin is used. Mowiol is acceptable.</li>
	<li>Place approximately 10-20 &#181;l of mounting medium on a slide. Invert coverslip (cell-side down) and mount coverslip gently, taking care to avoid introduction of air bubbles. Incubate slides for 24 hr (coverslip up) prior to imaging.</li>
	<li>Seal edges of coverslip with nail polish.</li>
</ol>
5. Experimental Setup
<ol>
	<li>Initiate required lasers and software. Initiate STED depletion laser at 100% power. Align STED laser, which in the case of commercial systems is often an automated procedure.</li>
	<li>Focus the sample, beginning with single stained control, on the microscope using eyepieces.</li>
</ol>
6. Optimization of Settings
<ol>
	<li>Scan the first channel and optimize laser power, excitation beam position and detector range. If possible, avoid a gain of &#62;100. Capture the image in confocal to optimize settings. Line and/or frame averaging will increase resolution. Check for pixel saturation. Note: Some saturation is acceptable in confocal as application of STED will reduce the intensity of emission. For STED, an optimal pixel size will be below 30 nm however better resolution will be obtained with smaller pixel sizes. The size of the region of interest being imaged will dictate the lower limit of pixel size. Smaller pixel sizes may increase photobleaching.
	<ol>
		<li>Apply STED depletion beam and capture image, starting with 50% depletion laser power. If an improvement in resolution is seen, more depletion laser power can be applied. At this stage, it may be necessary to adjust excitation laser power, line average, and/or gain.</li>
		<li>Apply time gating to reduce background (minimum 0.3 nsec). Adjust settings until an improvement in resolution over confocal can be seen. Resolution can be approximated by estimating full width half maximum (FWHM). &#160;This represents the distance at the half maximal intensity of a Gaussian peak created by drawing a line profile across the structure of interest, and is a widely used method of estimating resolution.</li>
		<li>Once the first channel is satisfactory, initiate a second sequence for sequential scanning. In general, it is best to scan the longer wavelength fluorophore first. Repeat step 6.1 on second channel.</li>
	</ol>
	</li>
	<li>Confirm lack of spectral overlap by imaging single stained controls with both scan sequences. Mild spectral overlap can be corrected using spectral un-mixing features in the software, however should be avoided wherever possible.</li>
</ol>
7. Image Acquisition
<ol>
	<li>Acquire images. For quantitative imaging, it is recommended to obtain at least 20 images/condition.&#160; The exact number, however, should be defined according to the experimental question in concert with a statistical approach such as sample size calculation. Save experiment.</li>
</ol>
8. Deconvolution
<ol>
	<li>Open file with deconvolution software or batch processor. Check parameters for each channel using software. Confirm each channel&#39;s excitation and emission spectra, STED depletion emission, and imaging direction (up or down, if the image is 3-dimensional) in particular.</li>
	<li>Deconvolve using the deconvolution wizard. Default settings are generally adequate, however signal to noise ratio (SNTR) will vary from fluorophore to fluorophore and will need to be determined for each channel and each experiment individually.</li>
</ol>

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The authors declare that they have no competing financial interests. Partial publication cost of this article was defrayed by Leica Microsystems, Inc.

The improvement in resolution over confocal will be somewhat dependent upon factors which cannot be controlled. These factors include minor aberrations in cover slip thickness and inconsistencies in mounting media. It is important to keep the temperature and humidity in the imaging room as consistent as possible, and the STED beam should be realigned approximately every 60 min. As mentioned in Procedures, use of VECTASHIELD mounting medium must be avoided, as this is not compatible with STED. In addition to which, one should always use #1.5 cover slips, and if available, use those which have been verified to a specific thickness.
One modification of the approach described here is to image additional channels in confocal, using fluorophores that emit at a longer wavelength than the STED beam. In this way, one can image up to four channels (two in confocal, two in STED). If taking this approach, however, the channels with fluorophores emitting above the STED depletion laser will need to be imaged first, as application of the STED beam will deplete photons in these channels. One advantage to this technique is the application of time gating, which will also improve resolution in confocal by eliminating emission from photons with short lifetimes34. In particular, the use of time gating, the timing of emission detectors to correspond with pulsed excitation STED, will decrease background fluorescence from reflection off coverslip glass when imaging close to it. Even in an experiment not suitable for STED, if using a pulsed excitation source, time gating can be a useful tool for improving resolution in confocal.
There are various modifications that can be utilized to improve resolution in STED. One is to decrease the size of the pinhole from the standard 1 Airy unit, although this will also decrease the amount of light reaching the sample. This can be compensated for by increasing laser power or gain. Another is to increase line average, which will increase the amount of information gathered for each photon, improving resolution. Again, however, this may be at the cost of photobleaching of the sample, so a balance will need to be struck between resolution and bleaching. Similarly, use of fluorescent proteins such as GFP will require careful optimization to avoid bleaching. This may be accomplished by decreasing STED laser power if necessary. Longer time points will also allow for greater photon recovery and reduce bleaching. Correction for photobleaching should be accounted for when analyzing live STED.
Of course, imaging in 3 dimensions in STED is also possible, and will also give an improvement over conventional confocal imaging. This is particularly true if it is done in combination with deconvolution, although care should be taken to correct for drift that occurs during imaging multiple planes in the z-axis. If using Huygens software to deconvolve, this correction is obtained using the "stabilize image" feature. Using this approach, resolution in the z-axis will be improved. This is a great improvement over conventional confocal imaging, which has poor axial resolution, and even over just STED itself, which also has relatively poor z-axis resolution. While acquiring multiple stacks in STED, care must be taken to avoid bleaching of the sample, and if necessary one can reduce line averaging or laser power intensity in order to do so. Again, it should be noted that if imaging other fluorophores that are not suitable for STED, application of the depletion beam in the first sequential scan would prevent emission from these channels. Therefore, a mixed STED/confocal approach (when using confocal scanning in channels that emit at a wavelength greater than 592 nm) will unfortunately not be suitable for 3D.
To summarize, we have chosen STED as an approach due to its relative ease of application and improvement in resolution over standard confocal imaging. For imaging the immune synapse, it has proven an effective and valuable technique that allows us to see details in F-actin architecture not possible at resolution over 200 nm. While many of these details seem subtle, they can have a profound effect on NK cell function. Thus, we are applying the latest nanoscopic imaging technology to deriving information that is critical for maintaining human health.

The immunological synapse is a complex milieu of signaling proteins and cytoskeletal elements. The cytolytic synapse was originally described as having a &#34;bulls-eye&#34; like structure with a ring of actin and adhesion molecules surrounding a central secretory domain1-4. However, we now know that it consists of microscopic domains of active signaling that require continuous dynamic cytoskeletal reorganization for function5-11. Much of the information that we now have about the synapse has been derived from microscopy, and immunologists have been early adopters of cutting-edge imaging technology.
One such novel technology is super-resolution microscopy. Conventional light microscopy is spatially limited by the diffraction barrier of light, which sets the lower limit of resolution for all fluorescence microscopy, including confocal, at approximately 200 nm. In recent years, several techniques have been developed that allow resolution below the diffraction barrier. These include stimulation emission depletion microscopy (STED), structured illumination microscopy (SIM), stochastically resolved microscopy (STORM), and photoactivatable light microscopy (PALM). These techniques have been reviewed in detail elsewhere12-15, but are outlined below. Subdiffraction limited resolution is generated in unique ways in each system. The selection of a super-resolution technique, therefore, should be dictated by the experiment and experimental system of interest.
STED super resolution is achieved using a high-intensity torroidal depletion beam that selectively &#34;silences&#34; fluorescence around each fluorophore of interest following excitation, resulting in subdiffraction limited fluorescence microscopy16-18. One advantage of STED is that image acquisition is rapid and requires relatively little post-processing. While dye selection is dictated by the spectral position of the depletion beam, which in the commercially available system is situated at 592 nm, several commercially available dyes are available that make combinations of two fluorophores possible. In addition, commonly used fluorescent reporters such as GFP can be imaged, making live cell experiments possible19,20.
We have previously used STED to identify and quantify regions of F-actin hypodensity that are utilized by NK cells for degranulation21,22. We propose that STED is a good choice for imaging the immune synapse due to its relatively flexible availability of fluorophores and superior improvement in resolution in the x-y axis. In addition, on the commercially available STED system utilized for these experiments, the use of a high-speed (12,000 Hz) resonance scanner allows for rapid acquisition of images with minimal damage to samples. Limited flexibility in dye selection is considered a disadvantage of STED12, however dual color STED is relatively straightforward with several commercially available fluorophores. The integration of STED with a laser scanning confocal microscope also allows for additional confocal imaging in combination with STED, so while STED is limited to two channels, additional structures can be imaged in confocal with resolution of approximately 200 nm (E. Mace, unpublished observations). While we describe the use of STED for imaging immune cells, this technology is applied to a variety of cell types, including neural cells, and for visualizing a variety of cell structures23-26.
SIM uses a different approach to generate subdiffraction-limited images. By visualizing known periodic excitation patterns, information can then be obtained about the unknown structure being studied following mathematical transformation27. This yields an increase in resolution to ~100 nm laterally28,29. The advantage of SIM is that it is compatible with all standard confocal dyes and probes, however the disadvantage is that it is much slower to acquire images and these require lengthy post-processing12. This also limits its use for live cell imaging.
Finally, super-resolution images can be generated by stochastic photo-switching of fluorophores. This approach is exploited in photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). By scanning multiple camera frames and localizing randomly activated molecules that are turned &#34;on&#34; and &#34;off&#34; over time, images with 20-30 nm resolution are generated from accumulated frames30-32 . The trade-off for this resolution is the time required to acquire images.
Here we show, in detail, the protocol for preparing and imaging dual color samples in STED. In this system, excitation is with a pulsed, tunable, white light laser. Due to the nature of the pulsed excitation beam, time gating of detection is made possible and further increases resolution. In addition, the system is equipped with gadolinium hybrid (HyD) detectors, which are more sensitive than conventional photomultiplier tubes, thus allowing for lower laser power requirements. The depletion beam for STED is applied continuously and is tuned to 592 nm, which will dictate the choice of dyes available for two color STED. Commonly used dye combinations generally include one excitable by 488 nm (such as Alexa Fluor 488, Oregon Green, DyLights green, or Chromeo 488) and one excitable by 458 nm (such as Pacific Orange or Horizon V500). Thus, while detection of the two dyes will be in a similar range (and both are accessible by the depletion laser), excitation will occur with different wavelengths. With a tunable white light laser and tunable detectors, maximizing signal while eliminating spectral overlap is made fairly easy. As such, we have had good success with combinations of commercially available dyes, such as Pacific Orange and Alexa Fluor 488 (used here).&#160; Our protocol is tailored towards and describes the evaluation of human NK cells as that represents the historical focus of our laboratory.&#160;We are specifically utilizing the NK92 cell line in this example as that is one we have regularly applied in our experimental work21,33.

<table border="1" cellpadding="0" cellspacing="0" style="width:602px;" width="602">
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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.

Clearly, a primary goal of super-resolution imaging will be an improvement over conventional confocal microscopy. However, there are some common pitfalls that may lead to suboptimal resolution. These require that each experiment be optimized individually. In our representative experiment, we are imaging the F-actin network in an NK cell activated by antibody bound to glass. Common causes of (and corrections for) a lack of improved resolution of STED over confocal are as follows:
<ol>
	<li>Under-sampling (Figure 1a). This may lead to graininess and loss of pixel information, as shown by poor resolution of F-actin filaments. Increased line or frame averaging can often correct this.</li>
	<li>Bleaching and/or over-sampling (Figure 1b). This may be caused by lengthy pixel dwell time as a result of excessive line averaging. Alternatively, it may be a result of over-scanning of the image prior to acquisition, including over-use of the depletion laser. This commonly results in hazy or fuzzy images. This can be corrected by scanning the field of interest only minimally before acquiring or, if possible, increasing laser scan speed. If the problem persists, the depletion laser power can be reduced.</li>
</ol>
By achieving the correct balance of pixel dwell time, excitation laser power, and depletion laser power, an image with improved resolution and sufficient information can be generated (Figure 1c). Resolution can be further improved by the use of deconvolution (Figure 1d). When acquisition is optimized, deconvolution will improve resolution both qualitatively and quantitatively and sub-100 nm resolution should be routinely attainable.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51100/51100fig1highres.jpg" src="/files/ftp_upload/51100/51100fig1.jpg" /> 
Figure 1. Optimization of acquisition and common pitfalls of STED imaging. NK92 cells were activated on anti-CD18 and -NKp30 coated glass for 20 min then fixed, permeabilized and stained for F-actin with Phalloidin Alexa Fluor 488. a) An example of loss of image information due to under-sampling. b) An example of loss of resolution due to bleaching/over-sampling c) conditions optimized d) optimized conditions lead to greater improvement in resolution with deconvolution. Scale bar = 5 &#956;m.

Watch this Scientific Journal Video about Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy at JoVE.com

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.

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 ...

visualization-immunological-synapse-dual-color-time-gated-stimulated

Research

JoVE Journal

Immunology and Infection

78.0K Views.  Texas Children's Hospital and Baylor College of Medicine.  Здесь мы проиллюстрируем протокол для работы с изображениями на двухцветной STED Наноскопии цитотоксический иммунный синапс киллеров воспроизводятся на стекле. С помощью этого метода мы получим разрешение суб-100 нм синапса белков и цитоскелета. 

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

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Facilitating the Analysis of Immunological Data with Visual Analytic Techniques

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the flexibility of a VA approach in the analysis of biological datasets. Examples of these datasets in immunology include flow cytometry, Luminex data, and genotyping (e.g., single nucleotide polymorphism) data. Contrary to the traditional information visualization approach, VA restores the analysis power in the hands of analyst by allowing the analyst to engage in real-time data exploration process. We selected the VA software called Tableau after evaluating several VA tools. Two types of analysis tasks analysis within and between datasets were demonstrated in the video presentation using an approach called paired analysis. Paired analysis, as defined in VA, is an analysis approach in which a VA tool expert works side-by-side with a domain expert during the analysis. The domain expert is the one who understands the significance of the data, and asks the questions that the collected data might address. The tool expert then creates visualizations to help find patterns in the data that might answer these questions. The short lag-time between the hypothesis generation and the rapid visual display of the data is the main advantage of a VA approach.

Visual analytics (VA) is a new approach of analyzing data interactively. In this video, we discuss the data overload problem brought on by high-throughput biological experiments, and propose VA as a solution to such problem. The video demonstrates analysis within and between immunological datasets using a VA tool called Tableau.

Visual analytics (VA) has emerged as a new way to analyze large dataset through interactive visual display. We demonstrated the utility and the ...

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFN&gamma; or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).

Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.

Macrophages are critical players in the innate  immune response to infectious challenge or injury, initiating the innate immune  response and directing ...

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans

In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections.

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

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 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.

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.

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 ...

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.

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...