Laboratuarımızda Araştırma Max von Pettenkofer Enstitüsü, Ludwig-Maximilians Üniversitesi Münih, Alman Araştırma Topluluğu (DFG, HI 1511/3-1) ve Bundesministerium für Bildung und Forschung (BMBF) &quot;Tıbbi Enfeksiyon Genomics&quot; inisiyatifi tarafından desteklenmiştir ( 0315834C).

1. LCV İzolasyon hazırlıkları <ol><li> Legionella pneumophila LCV izolasyon önce 5 mg / ml kloramfenikol (Cam) dört gün CYE Agar plaklarda gliserol stoklarından DsRed-Express 13,14 üreten dışarı Streak. 37 az bakteri inkübe ° C. </li><li> 1 x 10 üzerinden 7 Tohum D. GFP-calnexin veya 75 cm 2 doku kültürü RAW264.7 fare makrofajları üreten discoideum bir gün önce enfeksiyon matara. 20 ug / ml G418 D. için ile 10 ml HL5 orta kullanmak 23 ° C&#39;de discoideum ve amipler inkübe RAW264.7 makrofajlar% 10 FCS (ısıyla inaktive) ve% 1 glutamin ile desteklenmiş 10 ml RPMI 1640 ortamı, kullanımı ve 37 de hücrelerin büyümesi için ° C&#39;de ve% 5 CO2. Enfeksiyon ve örnek başına en az üç 75 cm 2 şişe (en az 6 x 10 7 hücre) kullanın. </li><li> L. ile bir gecede kültürü inoküle CYE plakadan pneumophila. AYE orta 3 ml ile 15 ml test tüpü alınve 5 ug / mL Cam. 0,1 bir OD 600 Nm vermek üzere, bir bakteri süspansiyonu 100 ul ile inoküle. 21-22 saat boyunca 37 ° C&#39;de bir havai rotasyon tekerlek üzerinde gecede kültür inkübe edin. </li></ol> 2. LCV İzolasyon <ol><li> D. orta değiştirin discoideum hücreleri aşağıdaki enfeksiyonu müdahale ederek antibiyotik, kaldırmak için. </li><li> Gecede kültür OD ölçün. Bakteriler 2 x 10 9 bakteri / ml karşılık ≥ 3 bir OD 600Nm, onların zirveye enfektivite ulaşmış olmalıdır. </li><li> L. yaklaşık 500 ul ilave edilerek hücreleri enfekte 10 ml HL5 ortamı (D. discoideum) ya da 10 ml RPMI 1640 (makrofajlar) büyüyen hücreler pneumophila bir gece kültüre. Bu 50 enfeksiyon çokluğu (İçişleri Bakanlığı) karşılık gelmektedir. Daha sonra, enfeksiyon 10 dakika boyunca 500 x g hızında hücreleri üzerinde bakteri santrifüjleme ile senkronize edilmektedir. Santrifüjleme takip ederD. bir kuluçka tarafından 25 ° C ve ° C&#39;de ve% 5 CO2, sırasıyla, 37 RAW264.7 makrofajlar azından discoideum hücreleri. 1 saat ile enfeksiyon süresi santrifüjleme adım içerir. </li><li> Enfeksiyondan sonra orta kaldırmak ve hücre dışı bakteriler kaldırmak için bir kez hücrelerin yıkayın. D. için buz SorC tampon kullanın discoideum ve RAW264.7 makrofajlar için buz gibi soğuk PBS. Her balona proteaz inhibitörleri (Roche) ile desteklenmiş 3 mL HS tampon, ekleme ve hücre kazıyıcı kullanarak hücreleri toplamak. 15 ml test tüpünde Havuzu gelen örnekleri. </li><li> Örnek kullanımı 3 mL plastik Luer-Lock şırınga ve paslanmaz çelik topu homojenizatörlerinin homojenizasyon için. Eğer buz üzerinde çalıştığından emin olun. Başlamadan önce, herhangi bir deterjan kirlenmesini önlemek ve hava kabarcıkları kurtulmak için buz HS tampon ile temizlemek için, damıtılmış su ile topu Homojenizatör yıkayın. 8 mikron boşluk topu kullanın. </li><li> Şırınga und içine ilk 3 ml doldurun th üzerine montee homojenizatör. Homojenizatör üzerinden ileri ve geri dokuz kez numune basın. -Homojen olmayan malzeme ile kirlenmesini önlemek için daha sonra şırınga ile değişin. Toplayın ve 15 ml test tüpünde homojenize örnek havuzundan ve mikroskopik analiz için 150 mikrolitre numune alır. Farklı bir örnek geçmeden önce demontaj ve topu Homojenizatör yıkayın. </li><li> 4 de buz üzerinde bir karıştırma cihazı üzerinde, 30 dakika süre ile ya da bir yükü-çıkrık (10-20 rpm) üzerinden% 2 veya CS FCS ile Homojenat bloke ° C. </li><li> Engelleme SidC (dilüsyon 1:3000) karşı veya LCV membran için özel olarak bağlayıcı herhangi bir diğer bakteriyel işaretleyici karşı bir yakınlık-saflaştırılmış primer antikor kullandıktan sonra. Homojenat eklemeden önce Vorteks antikor çözüm. 4 ° C&#39;de buz üzerinde bir çalkalayıcı içinde 1 saat süreyle ya da bir yükü-çıkrık (10-20 rpm) üzerine Örnek inkübe </li><li> Bu arada, Histodenz degrade hazırlamak. 15 ml degrade başına test tüpü ve bir örnek de üç 75 cm 2 şişeler kullanın. Köknarst, tüp% 35 Histodenz 5.75 ml ekleyin, ve ikinci, dikkatle üst üste% 10 Histodenz çözeltisi 5.75 mL ekleyin. Daha sonra dikkatli bir şekilde 1 saat boyunca aşağı doğru yatay borular yatıyordu. </li><li> Bloke edici reaktifin ve primer antikor, 4 pelet ° C, örnek olarak 15 dakika süre ile 600 x g&#39;de santrifüj ile Homojenizasyondan inkübasyondan sonra. Süpernatant atılır ve 1.5 mL HS tampon içinde pelletini. Taze bir 15 ml test tüpüne örnekleri aktarın ve daha sonra mikroskobik analiz için 40 ul örnek almak. </li><li> Sekonder antikor ile pelet inkübe - MACS keçi anti-tavşan IgG mikro boncuklar (seyreltme 01:25) -, bir karıştırma cihazı üzerinde, 30 dakika süre ile 4 ° C&#39;de Bu arada, MACS manyetik tutucu sütunlar koymak ve 0.5 mL HS tamponu ile bunları dengelemek. </li><li> Daha sonra, sütun örnekleri uygulanır. Sonraki mikroskopik analiz için akış-150 ul toplayın. 0,5 mL HS sütunu tamponu ile üç kez yıkanır. </li><li> T sütun çıkarınsquirting hemen 0.5 mL HS tamponu ile o mıknatıs ve eluat LCVs. Mikroskobik analizi için 20 mikrolitre numune alın. Immüno-manyetik ayırma ile saflaştırma 1. adımda başına verim bekleniyor × 10 7 D. enfekte discoideum hücreleri yaklaşık 1 × 10 6 bozulmadan hafif ticari araç, yaklaşık yedi kat flow-through 13 ile karşılaştırıldığında eluat zenginleşmiştir. </li><li> Dikey bir konuma Histodenz degrade tüpleri geri koyun ve dikkatle üstüne eluat yükleyin. 4, 1 saat boyunca 3350 x g&#39;de santrifüje tüpler ° C. </li><li> Santrifüj sonra uzun bir cam Pasteur pipeti kullanarak test tüpünün altından başlayarak 1.5 mL kesirler alır. Toplamda sekiz fraksiyonlar, borunun alt her toplamak. Hayır, görünen katmanları sürekli Histodenz degrade gözlenmektedir. LCVs en yüksek verimi genelde ~ 2 oranında kesir 4 bekleniyor başına 1 × 10 5 bozulmadan LCVs × 10 7amipler 13 ödenmiyor. LCVs Az miktarda sırasıyla, kesirler 3 ve 5 de mevcuttur. Böylece, saflaştırılmış hafif ticari araç toplam verimi ~ 1 × 10 × 10 6 7 başına 6 bozulmamış LCVs enfekte olan D. discoideum hücreler 13. LCVs ve geri kazanım oda sıcaklığında (RT) yapıldı ve LCVs birkaç saat boyunca stabil görünür edilebilir. </li></ol> 3. Toplanan numuneler mikroskobik analizi <ol><li> Poli-L-lizin kaplı lamelleri ile bir 24-kuyucuklu düz alt doku kültürü plakası hazırlayın. </li><li> LCV arıtma artı 24 de plaka içine her yoğunluk gradiyenti fraksiyonu 150 ul sırasında alınan her numune Pipeti. Yüksek Histodenz konsantrasyonu sulandırmak için her kuyuya 0.5 mL HS tampon ekleyin. Daha sonra, 4 azından 10 dakika süreyle 600 x g de santrifüj plakası ° C. Dikkatlice süpernatant kaldırmak ve RT 20 dk süreyle% 4 paraformaldehit (PFA) ile örnekleri düzeltmek. Tespit edildikten sonra örnekler iki kez yıkayın. BizeD. e SorC discoideum ve RAW264.7 makrofajlar için PBS. </li><li><ol class="lalpha"><li> D. ifade GFP-calnexin gelen örnekleri monte doğrudan örneğin Vectashield kullanılarak cam-slaytlar üzerinde LCV izolasyonu sırasında farklı adımlar alınan discoideum. </li><li> Oda sıcaklığında 10 dakika süre ile bloke çözeltisi (% 1 PBS içinde BSA) ile birlikte, LCV izolasyonu sırasında farklı aşamalarda alınan RAW264.7 makrofajlar, gelen numune inkübe. (; Tampon engelleme 1:1000 afiniteyle saflaştırılmış tavşan anti-SidC) LCVs leke ve RT de ıslak bir odasında 1s inkübe bir anti-SidC primer antikor kullanın. Daha sonra, PBS ile numuneler üç kez yıkayın. Oda sıcaklığında bir ıslak ve karanlık odaya 30 dakika süre ile, bir sekonder antikor (çözeltisi engelleme 1:200, örneğin anti-tavşan Cy5) ile inkübe edilir. Son olarak, PBS ile numuneler üç kez yıkayın ve örneğin Vectashield kullanılarak cam-slaytlar monte ederek. </li></ol></li><li> GFP-calnexin izole LCVs Morfolojisi, bütünlüğü ve miktarı ifade D. discoideum ya RAW264.7 makrofajlar yeterli bir filtre ile donatılmış bir floresans mikroskobu ile analiz edilir. </li></ol> 4. Temsilcisi Sonuçlar Immüno-afinite ayırma ve yoğunluk gradiyenti santrifüj LCV arınma kalite ve verim floresan mikroskobu (Şekil 1) takip edilebilir. Örnekleri arasında bir bakış D. gelen LCV izolasyonu sırasında toplanan discoideu m ya da makrofaj (Şekil 2) gösterilmektedir. L. Homojenat pneumophila-enfekte fagositler sağlam LCVs gösterir, aynı zamanda hücre artıkları ve bakterilerin hücre dışı bir sürü. Örnek peletleme sonra, görüntü, bazen, bir miktar daha yoğun Homojenat benzer. Bozulmamış LCVs sütun sopa gerektiğinden, flow-through çoğunlukla ekstraselüler bakteri ve hücre artıkları bulunur. Sütunundan Örnek elüt sonra, eluat, büyük miktarlarda bozulmadan LCVs içerir. Bizim ellerde, LCV purificati<html> D. için daha etkili olduğu görülmektedir makrofajlar için daha discoideum. D. discoideum patojen vakuol yuvarlak görünür ve izole LCVs verimi makrofajlar (Şekil 3) ile karşılaştırıldığında 10 kat daha fazladır. Bozulmamış makrofajlar farklı antikorları ile boyanan LVCs de daha düzensiz şekilli görünür. <img alt="Şekil 1" src="/files/ftp_upload/4118/4118fig1.jpg" /> LCV izolasyon Şekil 1. Şematik bakış. LCV membran münhasıran yerelleştirme, bakteriyel efektör protein SidC karşı bir yakınlık-saflaştırılmış antikor kullanarak immüno-manyetik ayırma ile LCVs A) İzolasyon. İkincil MACS mikro boncuk-coupled antikor ve bir mıknatıs hücre yıkıntıları arasından LCVs izole etmek için kullanılır. B) immüno-manyetik ayırma LCVs sonra daha da Histodenz yoğunluk gradyanı santrifüjleme vasıtasıyla saflandırılırlar. LCVs fraksiyonu 4 zenginleşmiştir. upload/4118/4118fig2.jpg &quot;/&gt; Şekil 2. Örnekleri LCV arıtma toplanmıştır. Homojenat, pelet, Görüntüler flow-through ve D eluat discoideum veya makrofajların tasvir edilmektedir. L. örnekleri göstermek pneumophila D. DsRed-Express (kırmızı) üreten GFP sinyali-calnexin (yeşil, üst panel) veya bir anti-SidC antikoru (siyan, alt panelini) ile boyanmış makrofajlarda üreten discoideum. Barlar, 10 mikron. <img alt="Şekil 3" src="/files/ftp_upload/4118/4118fig3.jpg" /> Şekil 3. D. gelen LCVs Saflaştırılmış discoideum veya makrofajların. Örnekleri L. Histodenz yoğunluk gradiyenti santrifüj sonrasında fraksiyonu 4 göstermek pneumophila (A) D. DsRed-Express (kırmızı) üreten GFP sinyali-calnexin (yeşil) üreten discoideum veya (B) bir anti-SidC antikoru (siyan) ile boyanmış makrofajlarda. Barlar, 10 mikron.

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Çıkar çatışması ilan etti.

Daha önce yayınlanmış yöntem tersine, bu protokolü iki aşamada, birinci bir immüno-manyetik bir yaklaşım tarafından LCVs ayrılması, ve ikinci olarak, yoğunluk gradyanı santrifüjle LCVs ile saflaştırma dayanmaktadır. LCV izolasyon kolayca floresan ile işaretlenmiş bakteri ve GFP üreten hücreleri veya antikor boyama da kullanılan floresan mikroskobu ile takip edilebilir. Burada açıklanan protokol yüksek saflıkta ile LCVs zenginleşmesine neden basit, düz ileri yöntemdir. Önemli olarak, homoj...

purification of pathogen vacuoles from legionella-infected phagocytes

Fırsatçı patojen Legionella pneumophila ayrıca, böylece ciddi pnömoni &quot;Lejyoner hastalığı&quot; 1 neden alveoler makrofajlarda çoğaltır bir amip dirençli bakteri vardır. Protozoon ve memeli fagositler, L. pneumophila belirli bir, çoğaltma-müsamahakar bölmesi, &quot;Legionella içeren vakuol&quot; (LCV) oluşturmak için korunmuş bir mekanizma kullanır. LCV oluşumu ev sahibi hücrelerin içine kadar 275 gibi &quot;efektör&quot; protein translocates bakteriyel ICM / tip IV Nokta salgılama sistemi (T4SS), gerektirir. Effektörler ana proteinler gibi yağlar işlemek ve salgı, endosome ve mitokondriyal organeller 2-4 ile iletişim kurarlar. LCVs oluşumu indirgemeci şekilde kavramak zor olan bir kompleks sağlam ve atıl işlemi temsil eder. Bütünleştirici bir yaklaşım kapsamlı yolu küresel bir analizini içeren LCV oluşumu, anlamak için gereklidirogen-konak faktörü etkileşimleri ve bunların zamansal ve mekansal dinamikleri. Bu amaca yönelik ilk adım olarak, bozulmadan LCVs saflaştırılmış ve proteomik ve lipidomics tarafından analiz edilmiştir. Bileşimi ve patojen-içeren vakuollerin oluşumu sıvı kromatografi-kitle spektrometresi ya akuple 2-D jel elektroforezi ile analiz proteomik tarafından araştırılmıştır. Sosyal toprak amip Diktiyostelyum diskodeyum ya da memeli fagositler ya izole Vakuoller Leishmania 5, Listeria 6, Mycobacterium 7, Rhodococcus 8, Salmonella 9 veya Legionella barındırmaktaydı. 10. Onlar örneğin elektron mikroskobu LCV morfolojisi, bütünlük ve saflık analiz gerektirir Ancak, bu çalışmalarda arıtma protokolü, zaman alıcı ve sıkıcı vardır. Ayrıca, bu protokoller için en patojen vakuol belirli özellikleri istismar yokrichment. Burada sunulan yöntemi D. istihdam ederek bu kısıtlamaların üstesinden Bir floresan LCV işaretleyici üreten discoideum ve bakteriyel efektör protein SidC, belirleyerek hangi seçici (PtdIns (4) P) 4-fosfat fosfatidilinositol için 3,11 bağlanarak LCV zarına çapalar. LCVs klasik bir Histodenz yoğunluk gradyanı santrifüjleme 12,13 (Şekil 1) tarafından, bir ikinci adımı takip SidC ve manyetik boncuklara bağlanan bir sekonder antikor, karşı afinitesi-saflaştırılmış primer antikor kullanılarak immüno-manyetik ayırma ile bir birinci adım zenginleştirilmiş . D. izole edilen hafif ticari araç bir proteom çalışma discoideum 13 öncesi LCV oluşumu karışmış edilmemiştir fagositik kesecikler, mitokondri, ER ve Golgi, yanı sıra çok sayıda GTPases ile ilişkili proteinler de dahil olmak üzere birden fazla 560 konak hücrenin protein, ortaya çıkardı. LCVs zenginleştirilmiş ve ile saflaştırılmışprotokolü daha mikroskobu (immunfloresan, elektron mikroskobu), biyokimyasal yöntemler (Western Blot) ve proteomik veya lipidomic yaklaşımlarla analiz edilebilir burada özetlenen.

Watch this Scientific Journal Video about Purification of Pathogen Vacuoles from Legionella-infected Phagocytes at JoVE.com

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Bu makale, bozulmamış izolasyonu ve saflaştırılması için bir yöntem açıklanmaktadır<em&gt; Legionella</emAmip ve makrofajlardan&gt; içeren vakuoller (LCVs). Iki adımlı bir protokol yoğunluk gradyanı santrifüj yoluyla bir bakteriyel LCV işaretleyici ve daha fazla arıtılmadan karşı bir antikor kullanılarak immüno-manyetik ayırma tarafından LCV zenginleştirme kapsamaktadır.

Patojen Vakuoller saflaştırılması gelen<em&gt; Legionella</em&gt;-Enfekte Fagositler

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

purification-of-pathogen-vacuoles-from-legionella-infected-phagocytes

Research

JoVE Journal

Immunology and Infection

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

12.4K Görüntüleme.  Ludwig-Maximilians-Universität. Bu makale, bozulmamış izolasyonu ve saflaştırılması için bir yöntem açıklanmaktadır Legionella içeren vakuoller (LCVs). Iki adımlı bir protokol yoğunluk gradyanı santrifüj yoluyla bir bakteriyel LCV işaretleyici ve daha fazla arıtılmadan karşı bir antikor kullanılarak immüno-manyetik ayırma tarafından LCV zenginleştirme kapsamaktadır.

Video: Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Bakteriyel Patojen İnsan Hücreleri Invasion

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

İmmünoloji ve Enfeksiyon

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Kan örneklerinden Bakteriyel Patojen Algılama ve Antimikrobiyal Direnç Testi için bir günlük iş akışı şeması

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Sıcak Sulu-fenol Ekstraksiyon tarafından Gram-negatif bakteriler arınma ve Lipopolisakkaritlerinin Görselleştirmesi

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Enfekte Hücre Kromatin gelen Gribi ribonükleoprotein Komplekslerinin Affinity Arıtma

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Fungal Patojen Fagositoz Canlı hücre video Mikroskopi

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Türler özgü Algılama Loop-aracılı İzotermal Amplifikasyon (LAMP) Tahliller<em&gt; Eimeria</em&gt; Tavuklar bulaştırmak ki

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA Arıtma Hücre içindeki dan Yetiştirilen<em&gt; Listeria monocytogenes</em&gt; Makrofaj

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

İnsan Nötrofil türetilmiş Dev Fagositler bir Roman ICSI&#39;nin Gelişimi ve Kimlik<em&gt; İn Vitro</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

Bir Maya İki hibrid Ekran kullanarak Olmayan ekilebilir Bitki Patojen bir Bakteriyel Efektör Fonksiyonu aydınlatmak

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

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.