Name: Video: Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy
Uploaded: 2014-03-24T20:00:00
Duration: 10 min
Description: 77.9K Görüntüleme. Texas Children's Hospital and Baylor College of Medicine. 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.

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.

Immünolojik sinaps proteinleri ve hücre iskeleti elemanları sinyalizasyon karmaşık bir ortam olduğunu. Sitolitik sinaps aslında merkezi bir salgı etki 1-4 çevreleyen aktin ve yapışma moleküllerinin bir halka ile yapı gibi bir &quot;boğa gözü&quot; sahip olarak nitelendirildi. Ancak, şimdi bu fonksiyonu 5-11 sürekli dinamik sitoskeletal örgütlenmesini gerektirir aktif sinyalizasyon mikroskobik etki oluştuğunu biliyoruz. Sinaps hakkında şu anda sahip bilgilerin çoğu mikroskop elde edilmiştir, ve immunolojistlerden üstün görüntüleme teknolojisi erken benimseyenler olmuştur. Böyle bir yeni teknoloji süper-çözünürlük mikroskopisidir. Geleneksel uzamsal ışık mikroskobu, yaklaşık 200 nm &#39;de dahil olmak üzere tüm konfokal floresan mikroskobu için çözünürlük alt sınırı, setleri ışık kırılma bariyer ile sınırlıdır. Son yıllarda, çeşitli teknikler geliştirmek olmuşturçözünürlüğü kırınım bariyerin altında izin d. Bu uyarım emisyon tükenmesi mikroskobu (STED&#39;in), yapılandırılmış aydınlatma mikroskobu (SIM), davranışsal çözüldü mikroskobu (FIRTINA) ve fotoaktıfleştırılebılır ışık mikroskobu (PALM) içerir. Bu teknikler yerlerinde detaylı olarak 12-15 gözden geçirilmiş, ancak aşağıda özetlenmiştir. Subdiffraction sınırlı çözünürlük her sistemde benzersiz şekilde oluşturulur. Süper çözünürlük tekniğinin seçimi, bu nedenle, ilgi konusu deney ve deneysel sistem tarafından dikte edilmelidir. STED süper çözünürlük subdiffraction sınırlı floresan mikroskobu 16-18 sonuçlanan uyarma sonrasında her bir ilgi florofor çevresinde seçici &quot;sessizliği&quot; floresan yüksek yoğunluklu torroidal tükenmesi ışını kullanılarak elde edilir. STED&#39;in bir avantajı görüntü elde etme, hızlı ve nispeten küçük bir post-processing gerektirir. Boya seçimi spec tarafından belirlenir daTicari olarak temin edilebilen sistemde 592 nm &#39;de yer almaktadır tükenmesi huzmesinin tral konumu, bir çok piyasada mevcut boyalar, iki florofor kombinasyonlar mümkün kılan mevcuttur. Buna ek olarak, GFP gibi yaygın olarak kullanılan floresan haberci 19,20 canlı hücre deneyleri mümkün hale görüntülenebilir. Daha önce degranülasyon 21,22 için NK hücreleri tarafından kullanılmaktadır F-aktin hipodansitedir bölgelerini belirlemek ve ölçmek için STED&#39;i kullandık. Biz STED&#39;in nedeniyle floroforlar ve xy ekseninde çözünürlükte üstün iyileşme nispeten esnek durumuna bağışıklık sinaps görüntüleme için iyi bir seçim olduğunu düşünmekteyiz. Buna ek olarak, bu deney için kullanılan, ticari olarak temin STED&#39;in sisteminde, yüksek hızlı (12,000 Hz) rezonans tarayıcının kullanılması örnekler en az hasar ile, görüntülerin elde edilmesi için hızlı sağlar. Boya seçimi sınırlı esneklik, STED&#39;in 12 bir dezavantaj olarak kabul edilirAncak çift renkli STED&#39;in piyasada bulunan çeşitli floroforlar nispeten basittir. STED iki kanal ile sınırlı iken, ek yapılar, yaklaşık 200 nm (E. Mace, yayınlanmamış gözlemler çözünürlükte konfokal görüntülü böylece bir lazer konfokal tarama mikroskobu ile STED&#39;in entegrasyonu da STED&#39;in ile kombinasyon halinde ilave confocal görüntüleme sağlar: .) Biz bağışıklık hücrelerini görüntülenmesi için STED&#39;in kullanımını tarif ederken, bu teknoloji, sinir hücreleri dahil olmak üzere hücre tipleri, çeşitli hücre ve yapıların çeşitli 23-26 görselleştirme için uygulanır. SIM subdiffraction sınırlı görüntüler oluşturmak için farklı bir yaklaşım kullanır. Bilinen periyodik uyarım desen ile görselleştirme, bilgi daha sonra, aşağıdaki matematiksel dönüşüm 27 çalışılmaktadır bilinmeyen yapısı elde edilebilir. Bu ~ 100 nm yanal 28,29 çözünürlüğe bir artış verir. SIM avantajı, bu is tüm standart konfokal boyalar ve probları ile uyumlu, ancak dezavantajı görüntüleri elde etmek için çok daha yavaş olduğunu ve bu uzun post-processing 12 ihtiyaç olmasıdır. Bu, aynı zamanda canlı hücre görüntüleme için kullanılmasını sınırlamaktadır. Son olarak, süper çözünürlüklü görüntüler Flüoroforlann stokastik foto-anahtarlama tarafından oluşturulmuş olabilir. Bu yaklaşım, fotoğraf aktive yerelleştirme mikroskobu (PALM) ve stokastik optik rekonstrüksiyon mikroskobu (FIRTINA) istismar edilmektedir. Birden fazla kamera kare tarama ve zamanla ve &quot;kapalı&quot;, &quot;açık&quot; rastgele aktif molekülleri lokalize ederek, 20-30 nm çözünürlükte görüntüler birikmiş çerçeveleri 30-32 üretilir. Bu çözünürlük için trade-off görüntüleri elde etmek için gerekli zamanı. Burada ayrıntılı olarak göstermek, hazırlama ve STED&#39;in çift renk örnekleri görüntüleme için protokol. Bu sistemde, atımlı bir uyarım, ayarlanabilir, beyaz ışık lazer ile. Doğası gereğidarbeli uyarma demetinin, algılama süresi yolluk mümkün kılan ve daha fazla artış çözünürlük edilir. Buna ek olarak, sistem, bu şekilde daha düşük bir lazer güç gereksinimleri için izin gadolinyum hibrid geleneksel foto-çoğaltıcı tüpler daha duyarlıdır (hyd) dedektörleri ile donatılmıştır. STED&#39;in için tükenmesi ışın sürekli olarak tatbik edilir ve iki renk STED&#39;in için mevcut boyaların seçimi dikte edecek 592 nm, ayarlanmıştır. Yaygın olarak kullanılan boya kombinasyonları genellikle (örneğin Alexa Fluor 488, Oregon Yeşil, yeşil DyLights veya Chromeo 488 gibi) 488 nm ile heyecanlı bir ve (örneğin Pasifik Turuncu veya Horizon V500 gibi) 458 nm ile heyecanlanan birini içerir. İki boyaların algılama benzer bir aralık içinde olacaktır (ve her ikisi de tükenme lazer tarafından erişilebilir) iken Böylece, farklı dalga boylarında uyarım ile ortaya çıkar. Bir ayarlanabilir beyaz ışık lazer ve ayarlanabilir dedektörleri ile, spektral örtüşme yok ederken sinyali maksimize oldukça kolay yapılır. Gibi, biz combinat ile iyi bir başarı olduBu tür Pacific Orange (burada kullanılan) Alexa Fluor 488 olarak ticari olarak temin edilebilen boya iyonları. Bizim protokol yönelik hazırlanmıştır ve bu bizim laboratuvar tarihsel odak temsil eder gibi insan NK hücrelerinin değerlendirilmesini açıklar. Biz düzenli olarak deneysel çalışmalar 21,33 olarak uygulamış biri olarak biz özellikle bu örnekte NK92 hücre hattını kullanıyor.

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visualization of the immunological synapse by dual color time-gated stimulated emission depletion (sted) nanoscopy

Doğal katil hücreler veya viral yönden enfekte tümörijenik hücrelerin lize etmek için sıkı bir şekilde düzenlenmiştir, ince ayarlı bir immünolojik sinaps (IS) oluşturur. Dinamik aktin tadilatı NK hücrelerin fonksiyonu ve IS oluşumu için çok önemlidir. Sinaps F-aktin Görüntüleme geleneksel ancak ışık kırılma sınır yaklaşık 200 nm, konfokal de dahil olmak üzere, flüoresan mikroskopi çözünürlüğünü sınırlandırır, konfokal mikroskopi kullanmıştır. Görüntüleme teknolojisindeki son gelişmeler subdiffraction sınırlı süper çözünürlük görüntüleme gelişmesini sağlamıştır. IS F-aktin mimarisi görselleştirmek için cam üzerinde reseptörü aktive NK hücreleri kalarak NK hücre sitotoksik sinaps özetlemek. Biz daha sonra iki-renkli uyarılmış emisyon tükenmesi mikroskobu (STED&#39;in) kullanarak ilgi görüntü proteinleri. Bu, sinaps &lt;80 nm çözünürlükte sonuçlanır. Bu yazıda numune hazırlama ve çift col kullanarak görüntüleri edinimi adımları açıklarveya NK F-aktin görselleştirmek STED&#39;in nanoscopy IS. Biz de Leica SP8 yazılım ve zaman-kapılı STED&#39;in kullanarak örnek edinme optimizasyonu göstermektedir. Son olarak, görüntülerin post-processing deconvolution için Huygens yazılımı kullanmak.

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.

Çift Renkli Zaman-kapılı Uyarılmış Emisyon tükenmesi (STED) Nanoscopy tarafından İmmünoloji Synapse görselleştirme

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

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

77.9K Görüntüleme.  Texas Children's Hospital and Baylor College of Medicine. 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.

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.

Kompleman tükenmesi Özel Hücre Populasyonlarının tükenmesi

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

İmmünoloji ve Enfeksiyon

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 Analitik Teknikleri ile İmmünolojik Veri Analizi kolaylaştırılması

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.

Farelerde Makrofajlar tükenmesi ve Sulandırma

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.

Makrofaj Kolesterol tükenmesi ve Fagositoz Üzerine Etkisi<em&gt; Cryptococcus neoformans</em

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.

Bir Cam destekli Planar çift katlı lipid Doğal Öldürücü Hücre İmmünoloji Synapse Süper çözünürlüklü Görüntüleme

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'.

Görüntüleme İmmünoloji Synapse Dynamics için bir Endotel Hücre Modeli Düzlemsel

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.

Akış Sitometresi Imaging kullanılarak insan sisteminde bağışıklık Synapse nitel ve nicel analiz

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.

Aktin ve Mikrotubul Cytoskeletons uyarılmış emisyonu tükenmesi (STED) mikroskopi kullanarak B-hücre bağışıklık sinaps, görselleştirme

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.

Immün SYNAPSE oluşumu sırasında B hücrelerinde organelle dinamikleri eğitimi

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

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long &#945;-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics DCAF

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Antijenik Peptid ve Fc-III Mimetics (DCAF) Çift Fonksiyonlu Konjuge tarafından Hedefli Antikor Engelleme

Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic ...