Bu çalışma ABD Tarım Dairesi, Tarımsal Araştırma Servisi tarafından desteklenmiştir. Bu yayındaki ticari adlar veya ticari ürünler belirli bilgi vermek amacıyla sadece belirtilen ve ABD Tarım Bakanlığı tarafından tavsiye veya onaylandığı anlamına gelmez. USDA bir fırsat eşitliği sağlayıcı ve işverendir.

1. Bakteriyel Colony Periferik Fringe <ol><li> X büyümek fastidiosa (Xf) Temecula yabani tip 22, pilG silme mutant PD2 orta agar plakaları 25 ° 28 ° üzerinde (daha önce kromozom tabanlı genetik tamamlama stratejisi 24 tarif kullanarak) (daha önce açıklanan silme stratejisi 23 kullanarak) Xf ΔpliG ve onun tamamlayıcı XfΔpliG -C 5-7 gün bekletilmiştir. </li><li> 15 dakika boyunca 121 ° C&#39;de (249 ° F) su içinde Otoklav selofan (1 x 1 cm2). , Selofan tek parça Pick up boş bir Petri kabı selofan bir köşesini dokunarak su tahliye dikkatle agar yüzeyi ve kuru hava% 15 üzerinde selofan yatıyordu. </li><li> Bireysel X Pick up aseptik agar plakaları agar yüzeyinin% 15 üst üste selofan bir sterilize levha üzerine yuvarlak kürdan ve spot hücreler steril ile fastidiosa koloniler. plaka inkübe2-3 gün 28 ° C &#39;de s. </li><li> 2X objektif lens ile diseksiyon mikroskobu ve 10X oküler lens kullanarak kolonilerin kenar morfolojisi inceleyin. kolonilerin etrafında çevresel saçak fotoğraf. </li></ol> 2. Mikroskopi ve mikroakışkan Akış Odaları <ol><li> Benzer fotoğraf litografik prosedürler kullanılarak Üretiyor mikroakışkan cihazlar önceden 5,18 nitelendirdi. Standart taşbaskı yöntemler 26 kullanarak ana silikon gofret bilgisayar destekli tasarım yazılımı ile dört paralel kanal tasarlayın. </li><li> polidimetilsiloksan (PDMS) ile silikon gofret ustadan mikroakışkan odaları oluşturun. silikon gofret ana üzerinde unpolymerized PDMS dökün ve 1 saat 60 ° C&#39;de onu tedavi. gofret PDMS kopya soyulabilir ve bir cam lamel aynı boyutu olarak 22 mm x 40 mm bir bıçak ile PDMS çoğaltma kırpın. </li><li> PDMS çoğaltma bir cam lamel (22 x 40 mm2) ve Microsc Açığa2 dakika 27 30 W hava plazmaya ope slayt (51 x 76 mm 2). Sandviç cam lamel ve mikro fl uidic odasını oluşturmak için cam mikroskop arasında PDMS gövde. </li><li> desenli kanal her bir ucunda PDMS bir delik (5.5 mm çap) delin. 12-20 cm uzunluğunda içine silikon kauçuk tüp kesti. PDMS yineleme kanalların her biri açılış ucuna silikon kauçuk hortumun bir ucu (dış çapı 5,1 mm, iç çap 2.1 mm, 0.8 mm duvar) ekleyin ve 1 saat boyunca 60 ° C&#39;de polimerize edilmemiş PDMS ile kapatın. </li><li> plastik luer konnektörleri dikenli sonuna kadar boru başka ucunu. Alüminyum folyo ile monte mikroakışkan odaları sarın ve 20 dakika boyunca her otoklav. </li><li> X bakteriler hücreleri toplayın kazıma yoluyla fastidiosa yabani tip mutant Xf ΔpliG ve tamamlayıcı XfΔpliG -C, tek aşılamak kullanarak PD2 orta plakalar döngüler. Hücre yoğunluğunu ayarlayınPD2 suyu 600 nm&#39;de 0.05 bir optik yoğunluğa kadar daha önce tarif edildiği gibi 23. 1 ml gaz geçirmez şırınga içine bakteriyel hücre çözeltisi toplayın. </li><li> ters bir mikroskop sahnede mikroakışkan cihaz monte edin. PD2 suyu içeren 5 ml gaz geçirmez şırınga bir giriş borusunu bağlayın. şırınga pompaları ile 5 ml Gaz Geçirmez şırınga takın. </li><li> Bir atık haznesine çıkış borusunu bağlayın. 0.2 ul dk orta akış hızını korumak -1 30 dakika sistemini stabilize etmek için. </li><li> bakteriyel hücre çözeltisi ihtiva eden bir 1 ml&#39;lik gaz geçirmez şırınga ile yandan giriş tüpleri bağlayın. Kanal ulaşılana kadar kauçuk tüp aracılığıyla bakteriyel hücre çözümü yıkayın. Bölmeden önce, görüntü yakalama, bağlanmamış hücreleri temizlemek için 0.2 ul dk&#39;lık bir araç akış hızı -1 30 dakika boyunca muhafaza edilir. </li><li> ışık kontrol etmek mikroskop alan düzeltilmiş bölümü altında mikroskop deklanşör monte edin. deklanşör co deklanşör bağlayınntrol sistemi ve bilgisayara kapı kontrol sistemini bağlayın. </li><li> mikroskop video portuna dijital bir kamera monte ve bilgisayara bağlayın. Menüden &quot;deklanşör&quot; işlevini seçin, time-lapse kayıt yazılımını çalıştırın ve yazılım ile çekim bağlantı kurmak için yazılımda otomatik olarak varsayılan olarak yüklenir deklanşör tanır. </li><li> yazılım otomatik olarak varsayılan yakalama aygıtı olarak dijital fotoğraf makinesi tanımak ve yazılım ile dijital kamera ile iletişim kurmak için time-lapse kayıt yazılımı menüden &quot;dijital fotoğraf makinesi&quot; işlevini seçin. </li><li> 20X faz-kontrast optikler kullanılarak kanalların birinde bakteri hücreleri bulup görüntü yakalama önce 40X objektif lens geçin. </li><li> mikroskop görüntüleri elde etmek için menüden varsayılan parametreleri kullanarak &quot;görüntü elde etme&quot; işlevini seçin, time-lapse kayıt yazılımı çalıştırın.Sonra, &quot;time-lapse Edinme&quot; fonksiyonunu açın ve 6-24 saat X&#39;in seğirmesi hareketliliğini gözlemlemek için gerekli deney bağlı süresince 30 sn 5,18,28 zaman aralığını ayarlamak fastidiosa 5,18,28. time-lapse kayıt başlatmak için &quot;Tamam&quot; a tıklayın. seçmek kayıt bittikten sonra bilgisayardaki hedef klasördeki görüntüleri yığını &quot;farklı kaydet&quot; menüden &quot;Yığın fonksiyonunu&quot; tıklayın. </li><li> birden fazla kanal için, 6 saat boyunca birinci kanaldan her 30 saniye zaman atlamalı çekim. hedef hücreleri bulmak için bir sonraki kanala mikroskop objektif lens taşıyın. Deney dört kanal kullanmak üzere ayarlanmış ise dört kanal sırayla her görüntü yakalamak için yukarıda anlatıldığı gibi zaman atlamalı fonksiyonunu tekrarlayın. sürece üç ardışık gün boyunca zaman atlamalı görüntü yakalama devam edin. Bütün deneyler oda sıcaklığında (23 ± 2 ° C) gerçekleştirilmiştir. </li><li> Bir içine zaman atlamalı görüntüleri derlemektime-lapse kayıt görüntüleme yazılımı kullanarak video dosyası. Run time-lapse kayıt yazılımı, menüden &quot;Yığın işlevi&quot; tıklatın ve bilgisayardan yığılmış dosyaları açmak için &quot;openstack işlevini&quot; seçeneğini seçin. </li><li> tüm görüntüleri seçip &quot;AVI&quot; çıkış biçimi seçerek, yığın modülünden &quot;filmi olun&quot; fonksiyonunu başlatın. bilgisayardaki Hedef klasördeki video dosyasını saklamak için &quot;farklı kaydet&quot; seçeneğini tıklayın. </li><li> bilgisayardaki hedef klasörün derlenen film seçin ve onları oynatmak. Sonra, oluşturulan video dosyaları bakteri hücrelerinin seğirmesi motilite aktivitesinin ortaya çıkan görselleştirme yoluyla tek hücre hareketliliğini gözlemlemek. </li></ol>

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Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

Bu çalışmada, biz X&#39;in hareket davranışını karakterize fastidiosa PilG mutant Xf ΔpilG ve yeni tasarlanmış çoklu paralel nano-kanal mikro fl uidic odalarında tamamlayıcı XfΔpilG- C suşları. Yeni tasarlanan mikro fl uidic odaları tek bir 50 mikron genişliğinde kanal 18 ile daha önceki tasarımlara göre genişliği 100 mikron nano-kanal ile en fazla dört paralel odaları olabilir. geliştirilmiş geniş nano-kanal medyanın akan ba...

Xylella fastidiosa Grapevine Pierce hastalığı (Vitis vinifera L.), 1,2, 3 dahil olmak üzere ekonomik olarak önemli bitkisel hastalıkların bir dizi neden olan bir Gram-negatif olmayan kamçılı, patojen bir bakteridir. Bu bakteri su ileten ksilem sınırlıdır gemiler. Asmanın Enfeksiyon ksilem gemi ve su stresi sonuçları ve beslenme yetersizlikleri 3 tıkanmasına neden olur. Başarılı kolonizasyon tesisi 3 geri kalanına enfeksiyon ilk siteden taşımak için bakterinin yeteneğine bağlıdır. X özelliği olan polar tip IV Pili 4 uzatılması, bağlanma ve geri çekme yoluyla flagellar bağımsız bakteriyel hareketi için bir araç motilitesini olan seğirmesi fastidiosa 5,6,7. Seğirmesi motilite lazer cımbız ve atomik kuvvet mikroskobu (AFM) 8,9,10 tarafından gözlenmiştir. Bu teknikler kullanılarak, twitching motiliteler N. tip IV pilus tarafından oluşturulan gonorrhoeae ve S. aeruginosa fl uorescently etiketleme pili ve mikroskopik hareketlerini yakalayarak ile karakterize edildi. Her iki yöntem bireysel bakterilerin yapışkan kuvveti ayrıntılı olmasına rağmen, prosedürler karmaşık ve zaman 9,10 alıcıdır. Mikro fl uidic odalar uzun mesafe, tek tek hücrelerin göçünü ve bakteri hücreleri 5,6 küçük agregalar gözlemlemek için kullanılmıştır. Bu odalar bir time-lapse görüntü kayıt sistemi 11,12,13,14 ile entegre bir tabak içinde bir microfabricated-nano-kanal olarak dizayn edilmiştir. Mikro uidic odacık cihazlar bakteri hareketi davranışı ve hücre-hücre etkileşimleri incelemek için çeşitli avantajlar sunar fl: (i) bir çok kanallı yetenekleri ile entegre bir platform sağlar; (Ii) hareketler ve bakteri nano ölçekli özellikleri tek hücre toplamlar inceleyebilirsiniz; (Iii) doğrudan m sağlarbakteriyel hücrelerin ve zaman atlamalı analizi icroscopic görüntü kaydı, (iv) bir mikro-ortamda bakterilerin tek tek ve / veya popülasyonlarının uzun süreli mekansal ve zamansal gözlemler sağlar; (V) bir kanalda kültür ortamının akış hızı hassas bir şekilde kontrol edilebilir ve kültür ortamında (vi) sadece çok küçük bir hacmi (1 mi), her bir deney için gereklidir. Son zamanlarda, mikro fl uidic akış sistemi çeşitli mikroçevrelerde 14,15,16 altında bakteri hücrelerinin davranışlarını araştırmak için istihdam edilmiştir. Yapışkanlık ve E. yüzey eki E. coli, 15 X fastidiosa 16 ve Acidovorax cam yüzeylere 14 mikro fl uidic odaları kullanılarak değerlendirildi citrulli. Acidovorax citrulli tipi IV pili aracılığıyla toplama ve biyo fi lm oluşumu 14 analiz edildi. A. Ayrıca, hareket citrulli akış c altında gözlenenşartlarından Tip IV pili kolonizasyon önemli roller oynayabileceğini göstermiştir ve A. yayıldı sap akış koşulları altında ksilem damarlarında citrulli. Pseudomonas aeruginosa ve X&#39;in seğirmesi motiliteler fastidiosa hücreleri başarılı bir şekilde mikrofabrike akış odasının 5,6,17 bir akışkan akımına karşı gözlendi. Tip IV pilus X. pilB ve pilQ mutantlar yoksun fastidiosa derinden mikro fl uidic cihazlar 5,6,18 içinde akış koşulları altında motilite seğirmesi hızını değiştirmek için bulunmuştur. Mikro fl uidic cihazlarda bakteri yapışma ve motilitesi üzerinde yapılan çalışmalar, mikro fl uidic odaları in vitro pili aracılı bakterilerin seğirmesi motilitesini ve göç analizi için özellikle uygun olduğunu göstermiştir. Bu sonuçlar hücre-hücre eki, agregasyonu ve kolonizasyonu içinde kolaylaştırır seğirmesi aracılı göç mekanizmasını açıklamakEv sahibi, sonuçta sistemik enfeksiyona yol. X. Pil-Chp operon fastidiosa kodlamak sinyal iletimi 20 yolaklarıyla pilG, pili, pilJ, hap, chpB ve chpC içerir. transmembran kemoreseptörlerin periplazmik etki kimyasal uyaranlara bağlamak ve sonuçta bakteriyel seğirmesi hareketliliğini kontrol etme sitoplazmik kısmı bir sinyal kaskad etkinleştirin. X&#39;in Pil-Chp operonunda fastidiosa, fosfo-geçiş proteini PilG Chey bir homologudur. E. E. coli ve P. aeruginosa, Chey kamçı motor proteinlerin 19, 21 ile etkileşim kemotaksis sistemlerinde yanıt düzenleyicisidir. X&#39;de virülansına doğru Pil-Chp operon katkıları olmasına rağmen fastidiosa çevresel sinyallere yanıt olarak ve X&#39;in ayarlı / motor tip IV Pili için, en son kemotaksis operonunda pilG rolü 20 incelenmiştir fastidiosa unc olduğunulear. X&#39;in seğirmesi hareketlilik aktivitesindeki kemotaksis regülatör pilG bir fikir aydınlatmak için fastidiosa, mikro fl uidic odası X&#39;in seğirmesi motilitesini değerlendirmek için kullanılır fastidiosa. X fastidiosa ve pilG bir silme mutantı Xf ΔpliG tamamlayıcı suşu XfΔpliG C ve in vitro olarak vahşi tip fenotipleri karşılaştırılması ile karakterize edilir. Sonuçlar X&#39;in seğirmesi motilite pilG rolünü vurgulamak fastidiosa.

<table>
	<tbody>
		<tr>
			<td>Biology materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>X. fastidiosa (Xf) Temecula wild type</td>
			<td></td>
			<td></td>
			<td>Costa, H. S., et al., 2004 22</td>
		</tr>
		<tr>
			<td>pilG deletion mutant Xf&#916;pliG</td>
			<td></td>
			<td></td>
			<td>Shi, X. Y., et al., 2007 26</td>
		</tr>
		<tr>
			<td>pilG complementary strain Xf&#916;pliG-C&#160;</td>
			<td></td>
			<td></td>
			<td>Davis, M. J., wt al. 1998 23</td>
		</tr>
		<tr>
			<td>Physical materials and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculating loops</td>
			<td>VWR international, Radnor, PA</td>
			<td>#22-363-607</td>
			<td>quantitative procedures such as bacterial collection</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Corporation</td>
			<td>#0002709226</td>
			<td>Sylgard 184 silicone Elastomeric Kits</td>
		</tr>
		<tr>
			<td>AmScope MD2000 digital camera</td>
			<td>AmScope, Irvine, CA</td>
			<td>SE305R-AZ-E</td>
			<td>Image, video recording and measurement&#160;</td>
		</tr>
		<tr>
			<td>Tubes line</td>
			<td>Edgewood, NY</td>
			<td>#T4300</td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Plastic luer connectors</td>
			<td>Edgewood, NY</td>
			<td></td>
			<td>Connected to the syringe and microfluidic chamber</td>
		</tr>
		<tr>
			<td>Syringe pumps</td>
			<td>Pico Plus, Harvard Apparatus, MA</td>
			<td>#702209</td>
			<td>The flow rate can be adjusted while the pump is running.</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Gastight, Hemilton Company, Reno, NV</td>
			<td>#1005</td>
			<td>Provide the flowing broth</td>
		</tr>
		<tr>
			<td>Inverted Olympus IMT-2 microscope</td>
			<td>Olympus</td>
			<td>IMT-2 FLuoro PHase</td>
			<td>Image observation and recording</td>
		</tr>
		<tr>
			<td>SPOT-RT digital camera</td>
			<td>Diagnostic Instruments, Inc., MI</td>
			<td>RT230</td>
			<td>Image, video recording and measurement</td>
		</tr>
		<tr>
			<td>Microscope Shutter</td>
			<td>The UNIBLITZ, US</td>
			<td>#LS2T2</td>
			<td>Control camera&#8217;s exposure time</td>
		</tr>
		<tr>
			<td>Microscope Shutter Control system</td>
			<td>The UNIBLITZ, US</td>
			<td>VCM-D1</td>
			<td>VCM-D1 Single Channel CE/UL/CSA Approved Shutter Driver</td>
		</tr>
		<tr>
			<td>MetaMorph Image software</td>
			<td>Universal Imaging Corp., PA</td>
			<td></td>
			<td>Real-time super-resolution image processing&#160;</td>
		</tr>
	</tbody>
</table>

visualization of twitching motility and characterization of the role of the pilg in xylella fastidiosa

Xylella fastidiosa bitkiler ekonomik açıdan önemli hastalıklara neden olan bir dizi, bir Gram-negatif olmayan kamçılı bir bakteridir. Seğirmesi motilitesi X sağlar fastidiosa uzun mesafe içi bitki hareketi ve kolonizasyonu için bir araç X patojenik doğru katkıda X&#39;in fastidiosa. seğirmesi motilite fastidiosa tip IV pili tarafından işletilmektedir. Xylella fastidiosa ve Tip IV pili pilG, sinyal transdüksiyon geçiş yolları ile ilişkili olan Pil-Chp operon&#39;u şifreleyen proteinler bir kemotaksis regülatörü tarafından düzenlenir. X&#39;in seğirmesi motilitesinde pilG rollerini aydınlatmak için fastidiosa, bir pilG -açıklı mutant Xf ΔpilG ve yerli pilG içeren tamamlayıcı suşu XfΔpilG- C geliştirilmiştir. Time-lapse görüntü kayıt sistemi ile entegre bir mikroakışkan odaları XfΔp içinde seğirmesi hareketliliğini gözlemlemek için kullanılanILG, XfΔpilG- C ve vahşi tip süzün. Bu kayıt sistemi kullanarak, uzun vadeli mekansal ve zamansal toplama gözlemlerini, tek tek hücrelerin ve seğirmesi hareketlilik yoluyla bakteri popülasyonlarının göç izin verir. X Mutant XfΔpliG seğirmesi eksik fenotip sergilenen oysa fastidiosa yabani tip ve tamamlayıcı XfΔpilG- C suşu, doğrudan mikroakışkan akış gözlerinden gözlenen tipik seğirmesi motilite özellikleri gösterdi. Bu çalışma pilG X. seğirmesi hareketliliğin artmasını göstermektedir fastidiosa. mikroakışkan akış odası seğirmesi hareketliliği gözlemlemek için bir araç olarak kullanılmaktadır.

Tip IV pilus-aracılı seğirmesi hareketlilik gösteren bir çevresel koloni saçak varlığı, X kolonileri gözlendi fastidiosa yabani tip ve tamamlayıcı Xf ΔpliG -C suşu (Şekil 1). Mutant XfΔpliG Ancak koloni çevresi etrafında bir saçak (Şekil 1) sergilemedi. Nano-mikroakışkan akış odalarına bakteri hücrelerinin Time-lapse görüntüleme seğirmesi motilite hem vahşi tip X gözlen...

Watch this Scientific Journal Video about Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa at JoVE.com

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Bu çalışmada, nano-mikroakışkan akış odası görselleştirmek ve işlevsel Xylella fastidiosa, dedikodu olarak Pierce hastalığa neden olan bir bakterinin seğirmesi motilitesini karakterize etmek kullanılmıştır.

Rolü seğirmesi Motilitesi görselleştirme ve Karakterizasyonu<em&gt; PilG</em&gt; &#39;de<em&gt; Xylella fastidiosa</em

Xylella fastidiosa is a Gram-negative non-flagellated bacterium that causes a number of economically important diseases of plants. The twitching motility ...

The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells and populations.Demonstrating this procedure will be Dr.Xiangyang Shi, a post-doc from my laboratory.Using a standardized deletion strategy, grow Temecula wild-type Xylella fastidiosa, grow pilG deletion mutants, and grow the complimentary strain all on PD2 medium agar plates.Incubate the bacteria at 28

visualization-twitching-motility-characterization-role-pilg-xylella

Research

JoVE Journal

Immunology and Infection

Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

10.6K Görüntüleme.  University of California, Davis. The over goal of this protocol is to functionally characterize the genes responsbile for the twitching motility of bacteria using a nano microfluidic flow chamber integrated with a time-lapse image recording system.This method can help answer key questions about a bacteria phenotype and associated gene function, such as the twitching motility of bacteria.The main advantage of this technique is that it provides a special and a tempered observation for aggregation migration of individual cells ...

Video: Visualization of Twitching Motility and Characterization of the Role of the PilG in Xylella fastidiosa

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. The NALT are two parallel bell-shaped structures located in the nasal passages above the hard palate, and are usually considered to be secondary components of the mucosal-associated lymphoid system4-6. Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells4,7,8. These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages9,10. Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens7. While NALT disappear in humans by the age of two years, the Waldeyer's Ring and similarly structured lymphatic organs continue to persist throughout life6. In contrast to humans, mice retain NALT throughout life, thus providing a convenient animal model for the study of immune responses originating within the nasal sinuses11.
Cultures of single-cell suspensions of NALT are not practical due to low yields of mononuclear cells. However, NALT biology can be examined by ex vivo culturing of the intact organ, and this method has the additional advantage of maintaining the natural tissue structure. For in vivo studies, genetic knockout models presenting defects limited to NALT are not currently available due to a poor understanding of the developmental pathway. For example, while lymphotoxin-&alpha; knockout mice have atrophied NALT, the Peyer's patches, peripheral lymph nodes, follicular dendritic cells and other lymphoid tissues are also altered in these genetically manipulated mice12,13. As an alternative to gene knockout mice, surgical ablation permanently eliminates NALT from the nasal passage without affecting other tissues. The resulting mouse model has been used to establish relationships between NALT and immune responses to vaccines1,3. Serial collection of serum, saliva, nasal washes and vaginal secretions is necessary for establishing the basis of host responses to vaccination, while immune responses originating directly from NALT can be confirmed by tissue culture. The following procedures outline the surgeries, tissue culture and sample collection necessary to examine local and systemic humoral immune responses to intranasal (IN) vaccination.

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue NALT in Mouse Responses to Vaccines

Methods to examine contributions of the nasopharyngeal-associated lymphoreticular tissues (NALT) to nasal and systemic immune responses of mice to intranasal vaccines are described. We demonstrate a surgical procedure to establish a NALT-dependent mouse model and ex vivo cultures of extracted NALT.

Aşılar Fare yanıtlar içinde Nazofaringeal ilişkili lenforetiküler Doku (NALT) Rolü incelenmesi

The nasopharyngeal-associated lymphoreticular tissues (NALT) found in humans, rodents, and other mammals, contribute to immunity in the nasal sinuses1-3. ...

İmmünoloji ve Enfeksiyon

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Phagokinetic Parça Motilite Testi sayesinde Hücre Göç Kantitatif Değerlendirilmesi

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Nitel ve Nicel Saptanması için Tahliller ve Protein Antimikrobiyal karakterizasyonu

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Kan işleme için hızlı bir yöntem insan kanı plazma peptid düzeylerinin veriminin arttırılması için

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.

This report describes protocols for characterizing interactions between bacterial outer membrane proteins and the human complement regulator vitronectin. The protocols can be used to study the binding reactions and biological function of vitronectin in any bacterial species.

Vitronectin bakteriyel yapışma ve Serum direnç rolü eğitim için deneyleri

Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Yalıtım, karakterizasyonu ve obezite ile ilgili iltihap tarafından etkilenen dokular makrofajlar arıtma

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Isolation and Characterization of Extracellular Vesicles Produced by Iron-limited Mycobacteria

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) containing immunologically active molecules. Knowledge regarding the molecular mechanisms of vesicle biogenesis, the content of the vesicles, and their functions at the pathogen-host interface is very limited. Addressing these questions requires rigorous procedures for isolation, purification, and validation of EVs. Previously, vesicle production was found to be enhanced when M. tuberculosis was exposed to iron restriction, a condition encountered by Mtb in the host environment. Presented here is a complete and detailed protocol to isolate and purify EVs from iron-deficient mycobacteria. Quantitative and qualitative methods are applied to validate purified EVs.

Mycobacterium tuberculosis shows increased production and release of extracellular vesicles in response to low iron conditions. This work details a protocol for generating low iron conditions and methods for the purification and characterization of mycobacterial extracellular vesicles released in response to iron deficiency.

Demir sınırlı Mycobacteria tarafından üretilen Ekstrasellüler Veziküllerin İzolasyon ve Karakterizasyonu

Mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, naturally release extracellular vesicles (EVs) ...

Visualization, Quantification, and Mapping of Immune Cell Populations in the Tumor Microenvironment

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

Here, we describe a simple and accessible strategy for visualizing, quantifying, and mapping immune cells in formalin-fixed paraffin-embedded tumor tissue sections. This methodology combines existing imaging and digital analysis techniques with the purpose of expanding the multiplexing capability and multiparameter analysis of imaging assays.

Tümör Mikroortamında Bağışıklık Hücre Popülasyonlarının Görselleştirilmesi, Ölçülmesi ve Haritalanması

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Floresan Antibiyotik Probları Kullanılarak Bakteriyel Direncin Görüntülenmesi

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Characterization of Immune Cells and Proinflammatory Mediators in the Pulmonary Environment

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be altered by severe brain injury. This is evidenced by the fact that pneumonia is a major cause of mortality in patients who have suffered from ischemic stroke. The goal of this protocol is to describe the use of multicolor flow cytometric analysis to identify 13 types of immune cells in the lungs of mice, including alveolar macrophages, interstitial macrophages, CD103+ or CD11b+ dendritic cells (DCs), plasmacytoid DCs, eosinophils, monocytes/monocyte-derived cells, neutrophils, lymphoid-derived&#160; T and B cells, NK cells, and NKT cells, following ischemic stroke induction by transient middle cerebral artery occlusion. Moreover, we describe the preparation of lung homogenates using a bead homogenization method, to determine the expression levels of 13 different cytokines or chemokines simultaneously by multiplex bead arrays coupled with flow cytometric analysis. This protocol can also be used to investigate the pulmonary immune response in other disease settings, such as infectious lung disease or allergic disease.

This protocol describes the use of flow cytometry to identify the changes in immune cell composition, cytokine profile, and chemokine profile in the pulmonary environment following transient middle cerebral artery occlusion, a murine model of ischemic stroke.

Pulmoner Ortamda İmmün Hücrelerin ve Proinflamatuar Mediatörlerin Karakterizasyonu

Immune cell expansion, activation, and trafficking to the lungs, which are controlled by the expression of multiple cytokines and chemokines, may be ...