Name: rna purification from intracellularly grown listeria monocytogenes in macrophage cells
Uploaded: 2016-06-04T14:00:00.000+00:00
Duration: 10 min 42 s
Description: Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.

Not: tüm deney sırasında, makrofaj hücreleri,% 5 CO2 basınçlı hava kuluçka makinesi içinde 37 ° C sıcaklıkta kuluçkaya yatırılır ve yalnızca bir sınıf biyolojik güvenlik kabininde yapıldığı deneysel manipülasyonlar için kuluçka alınır. L. ile çalışma monocytogenes bakteri biyolojik güvenlik seviyesine 2 düzenlemelerine göre olduğunu. 1. Hücre Hazırlama ve bakteri kökenli enfeksiyon (Gün 1 ve 2) <ol><li> 1.gün <ol><li> 30 mi BMDM + Pen-Strep ortamı (Tablo 2) ve 145 mm tabak Tohum 2.0 x 10 7, kemik iliği kaynaklı makrofaj hücreleri (BMDM). Bakteriyel suşların her biri için tohum 3 tabak analiz edilmesi. % 5 CO2 basınçlı hava kuluçka makinesi içinde 37 ° C&#39;de gece boyunca inkübe edilir. </li><li> Vahşi tip (WT) L. bir gece boyunca bakteri kültürü Başlangıç Standart bir inkübatörde 30 ° C&#39;de, beyin kalp infüzyon (BHI) ortam 10 ml streyn monocytogenes 10403S. Yeri kültür tüpleri sallayarak olmadan eğimli. Not:Bu büyüme koşulları etkili bir şekilde enfeksiyonu teşvik flagella genlerin ekspresyonunu yukarı-regüle. </li></ol></li><li> 2. gün <ol><li> 37 ° C&#39;de ön-Sıcak BMDM (Pen-Strep antibiyotiksiz) ortamı (Tablo 2) ve fosfat tamponlu tuz (PBS). </li><li> antibiyotik kaldırmak için önceden ısıtılmış PBS, 25 ml ile iki kez makrofaj tek tabaka yıkayın. 30 ml taze BMDM orta ekleyin. </li><li> 1 dakika boyunca&gt; 14,000 xg&#39;de santrifüj bakteri süpernatant atılması ve pipetleme PBS 1.5 ml yavaşça bakteri resuspending PBS ile gece boyunca bakteri kültürü 1.5 mL yıkanır. İki kez tekrarlayın. </li><li> Vahşi tip L yıkanmış bir gece boyunca bir kültürü, 0.5 ml her makrofaj plaka Infect monocytogenes. Birden fazla plakaları kullanıyorsanız, ayrı her plaka 15 dakika bulaştırmak. Bu zaman aralığı enfeksiyonu sonunda tek tek her plaka hasat sağlayacaktır. Not: enfeksiyon (MOI) seri seyreltme kaplama ile deneye tabi tutulurBakteri kültürünün s, 37 ° C&#39;de plakalar 24 saat inkübe edildikten sonra, BHI agar plakaları ve sayma koloni oluşturan birim (CFU) ile enfeksiyonu için kullanılmıştır. WT L. 0.5 ml kullanılarak monocytogenes ~ 100 bir İçişleri Bakanlığı içinde 10403S sonuçlar (makrofaj hücre başına 100 bakteri CFU) süzün. hücre içi büyüme hatalı bakteriyel mutantlann analiz edildiğinde, daha yüksek bir MOI dikkate alınmalıdır. </li><li> 37 ° C&#39;de 0.5 saat inkübe edildikten sonra, serbest bakterileri uzaklaştırmak için PBS ile iki kez enfekte hücreleri yıkayın ve 30 ml önceden ısıtılmış BMDM ortamı ilave edin. Ekli bakteriler sonraki 0.5 saat boyunca içselleştireceklerdir. </li><li> hücre-dışı bakterileri öldürmek için: (50 ug / ml bir nihai konsantrasyon elde etmek 1000 1) 1 saat sonra enfeksiyondan, gentamisin 30 ul ekle. </li><li> Bir vakum çıkış portu ile bir toplama sıvı şişesi üzerinde bir filtre kafasını koyarak filtre aparatı monte edin. Sonra silindir bir huni tarafından izlenen bir filtre kafasına bir filtre (0.45 um), koyun ve bir metal c farklı bölümlerini sabitleyinlamba. buz soğukluğunda RNaz içermeyen su hazırlayın. </li><li> aşağıdaki şekilde enfekte olmuş hücrelerden 6 saat sonrası enfeksiyon, hasat bakterileri. tek tek her plaka davranın. Not: Genellikle, L. monocytogenes makrofaj hücrelerinin enfeksiyon 6 saat boyunca büyüme 1-log tamamlar. kısa inkübasyon süreleri önemli ölçüde daha düşük bakteri yüklerini neden olabilir ise uzun inkubasyon, bakteriyel aşırı çoğalma ve makrofajların hücre ölümüne neden olabilir. MOI, enfeksiyon süresi optimal şartlara ulaşmak için değiştirilebilir. <ol><li> bir kez PBS ile enfekte hücreleri yıkanır. makrofaj hücreleri lize etmek için buz-soğuğu RNaz içermeyen su 20 ml ekleyin. Bir hücre kazıyıcı kullanarak, hızlı ama dikkatli plaka kapalı hücreleri kazıyın. </li><li> 50 ml konik tüp lize hücreleri toplamak. 30 saniye vorteksleyin. 4 ° C&#39;de 3 dakika boyunca 800 x g&#39;de santrifüjleyin. </li><li> bir vakum sistemi kullanılarak filtre aparatı ile süpernatant geçirin. coni ml filtre rulo ve hızlı bir şekilde 15 transfer, cımbız kullanarakcal tüp. sıvı azot içinde filtreler ile tüpler Yapış-dondurma. </li><li> 1.2.7 de tarif edildiği gibi, aşağıdaki örnek için yeni bir filtre ile filtrasyon aparatı birleştirin. </li><li> Bir sonraki gün için -80 ° C&#39;de donduruldu filtreleri saklayın. Seçenek olarak ise, bir nükleik asit çıkarma, doğrudan devam edin. </li></ol></li></ol></li></ol> 2. Nükleik Asitler Ekstraksiyon (Gün 3) Not: Bir çeker ocak içinde, fenol ve kloroform çözümler Bütün müdahaleler gerçekleştirin. <ol><li> 1 asidifiye fenol karışımı: kloroform, her bir örnek için 400 ul 1 hazırlayın. Ayrı tüplerde karıştırın ve daha sonra aspirasyon ile ortaya çıkan sulu tabaka çekilme. % 10 SDS, 40 ul ekle. </li><li> Soğuk tutmak için buz üzerinde filtre içeren tüpler çözülme. Her bir filtre ihtiva eden bir tüpe asetat-EDTA (AE) tampon maddesi (Tablo 2) 650 ul ilave edin. </li><li> mümkün olduğunca çabuk aşağıdaki adımları uygulayın. <ol><li> Şiddetle bu yüzden filtreyi içeren tüp vorteksFiltre tüpün çevresine whisks ve tampon tamamen filtreyi yıkar. Her zaman buz üzerinde geri koyarak tüp soğuk tutmak. Not: Tam filtre kapalı bakteri yıkamak için ters ise ayrıca tüp vorteks için gerekli olabilir. </li><li> Tüm borular için tekrarlayın. Spin-aşağı kısa bir süre süspansiyon (120 xg&#39;de 1 dakika) işlemi sonunda yararlı olabilir. </li></ol></li><li> SDS ve (2.1 hazırlandı) fenol / kloroform karışımı ihtiva eden bir 1.5 ml mikrosantrifüj tüpüne bakteri içeren tampon aktarın. Tüm borular için tekrarlayın. Filtreden kalan tampon almak için tekrar filtre ihtiva eden tüpler (120 xg&#39;de 1 dakika) döndürmek için gerekli olabilir. </li><li> 10 dakika süre ile tam hızda her 1.5 mi, bir multi-tüp vorteks cihazı tüpler ve vorteks yerleştirin. </li><li> 10 dakika boyunca 65 ° C ısıya bloğunda inkübe edin. 5 dakika boyunca maksimum hızda (&gt; 14.000 xg) santrifüj. </li><li> Her t sulu tabakayı (yaklaşık 400 ul) aktarın3 M sodyum asetat (pH 5.2) ve 1.0 ml% 100 etanol içinde 40 ul ihtiva eden yeni bir 1.5 ml&#39;lik tübe Ube. iyice her tüp Vortex. Not: Glikojen çöktürülmüş pelet görselleştirme artırmak için bu aşamada ilave edilebilir. </li><li> 1 saat boyunca -80 ° C&#39;de inkübe edin. Seçenek olarak ise, Cı gece boyunca -20 ° örnekleri inkübe edin. Daha sonra, en yüksek hızda (&gt; 14,000 xg), 20 dakika boyunca 4 ° C &#39;de numuneler santrifüj. </li><li> Dikkatle her tüpten etanol (üst katman) aspire. iyice her bir örnek ve vorteks 500 ul soğuk% 70 etanol ekleyin. maksimum hıza (&gt; 1 4000 xg), 20 dakika boyunca 4 ° C&#39;de santrifüje. </li><li> Dikkatle her tüpten etanol aspire. Resim ısı ile bir vakum buharlaştırıcı kullanılarak yaklaşık 2 dakika boyunca numune kurutun. numuneler aşırı kurutmayın. </li><li> Her bir örnek için 25 mL RNaz içermeyen su ilave edilir. 20 dakika oda sıcaklığında inkübe edin. Dikkatle girdap ve spin-aşağı. </li><li> t RNA konsantrasyonu ölçmekBir mikrohacim UV-Vis spektrofotometre kullanarak o örnekler. Plaka başına nükleik asitlerin 1 ug - yaklaşık 0.5 ayıklamak için bekliyoruz. Not: teknik çoğaltır bu aşamada birleştirilebilir. </li></ol> 3. DNaz Tedavi <ol><li> Mikrofüj tüpüne Tablo 1 &#39;e göre bir reaksiyon ayarlayın. 45 dakika boyunca 37 ° C&#39;de inkübe edin. 450 mL RNaz içermeyen su ve maksimum hızı (&gt; 14,000 xg) 2 dakika süreyle santrifüjlenerek fenol-kloroform-IAA karışımı (Tablo 2), ayrı fazlar 500 ul ekle. </li><li> Yeni bir tüpe Sulu tabaka transferi ve maksimum hızı (&gt; 14,000 xg) 2 dakika santrifüjleme ile 500 ul kloroform-IAA karışımı (Tablo 2), girdap ve ayrı fazlar ekleyin. </li><li> yeni bir tüpe Sulu tabaka transferi ve 1 mL etanol ve 3 M sodyum asetat (pH 5.2) 50 ul ekle. Vortex. Not: Glikojen çöktürülmüş pelet görselleştirme artırmak için bu aşamada ilave edilebilir. </li><li&gt; -80 ° C de 1 saat süreyle inkübe edin. Seçenek olarak ise, Cı gece boyunca -20 ° örnekleri inkübe edin. maksimum hızda 20 dakika süreyle 4 ° C&#39;de santrifüje. </li><li> Dikkatle her tüpten etanol aspire. iyice her bir örnek ve vorteks 500 ul soğuk% 70 etanol ekleyin. en yüksek hızda 20 dakika süreyle 4 ° C&#39;de santrifüje. Dikkatle her tüpten etanol aspire. </li><li> Resim ısı ile bir vakum buharlaştırıcı kullanılarak yaklaşık 2 dakika boyunca numune kurutun. numuneler aşırı kurutmayın. </li><li> Oda sıcaklığına girdap ve dönme, yavaşlama, 2 dakika boyunca inkübe RNaz içermeyen su 12 ul ekle. Numuneler, saflaştırılmış RNA içeren buz üzerinde saklayın. Seçenek olarak ise, RNA çözülür RNase içermeyen H2O 0.1 mM EDTA ve TE tamponu (10 mM Tris, 1 mM EDTA) ile. </li><li> Bir mikrohacim UV-Vis spektrofotometre kullanılarak RNA konsantrasyonları ölçün. (Teknik çoğaltır birleştirilir ise) numune başına RNA ~ 100 ng bekliyoruz. Not: RNA çıkarılan bu protokole göre fo uygundurBirden tekniklerle r gen transkripsiyonu analizi. Biz genellikle L. incelemek için RT-qPCR analizi istihdam makrofaj hücreleri 1-4 enfeksiyon sırasında gen transkripsiyonunu monocytogenes. </li></ol>

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The authors have nothing to disclose.

Burada açıklanan protokol L. bakteriyel RNA izolasyonu için optimize edilmiş bir yöntemle temsil makrofaj hücrelerinde hücre büyüyen bakterilerin monocytogenes. Bu protokol, hücre ayırıcı liziz göre ve bakteriyel RNA zenginleştirilmesi için iki ana adımları içerir: santrifüj ve süzme ile bakterilerin hızlı bir koleksiyonunu kullanarak makrofaj çekirdekler çöktürülür. Bu adımlar, standart RNA ekstraksiyon prosedürü izlemektedir. Bu protokol, Lısterıal RNA saflaştırma ...

Hücre içi bakteriyel patojenler ─ bulaşıcı hastalıklar ve büyüyen insan konak iç hücrelerini üretebilen neden olan bakteriler ─ dünya çapında önemli bir sağlık sorunu 5 vardır. istila ve bir memeli hücre içinde çoğaltmak için, hücre içi patojenler sofistike virülans mekanizmaları ve faktörleri edindik. bu mekanizmalar bir hastalığa neden yeteneği temel olmakla birlikte, biz onların düzenlenmesi ve dinamikleri hakkında çok az şey biliyoruz. sıvı ortamda yetişen bakteri geni ekspresyonunun konak hücreleri içinde, gerçek bir ortam yansıtamıyacağından, hücre içi niş yetiştirilen bakteri analiz transcriptome için giderek artan bir ihtiyaç vardır. Bu tür analizler ev sahibi tarafından tetiklenen spesifik bakteriyel uyarlamalar deşifre sağlayacak ve tedavi tasarımı için yeni hedefler belirlemeye yardımcı olacaktır. memeli RNA da bakteriyel RNA sayıca üstün beri hücre yetiştirilen bakterilerin Transcriptome analizi son derece zorluen az on kat. Bu yazıda, fare makrofaj hücreleri içinde büyüyen Listeria monocytogenes bakterilerden bakteriyel RNA izole etmek için deneysel bir yöntem açıklanmaktadır. ekstre RNA, hücre içi uyarlama ve RT-PCR, RNA-DİZ, mikrodizi ve diğer melezleştirme tabanlı teknolojiler gibi transkripsiyon analizleri çeşitli tekniklerle, patojenik bakterilerin hastalık oluşturma mekanizmaları incelemek için kullanılabilir. L. monocytogenes insanlarda listeriosis etkeni öncelikle immün sistemi baskılanmış bireyler, yaşlılar ve hamile kadınlar 6 hedefleyen klinik bulgularla seyreden bir hastalıktır. O 7 çalışma evsahibi-patojen etkileşimlerinin bir model olarak yıllardır kullanılmaktadır memeli hücrelerinin geniş bir dizi işgal bir Gram-pozitif fakültatif hücre içi patojendir. istila etmesine, bu t kaçmasına gereken bir vakuol ya da (fagositik hücreler durumunda), bir fagozomun, başlangıçta bulunduğuo çoğaltmak için hücre sitoplazmada ev sahipliği. Muhtelif virülans faktörlerinin çıkış işlemi, temel olarak, gözenek-oluşturucu hemolisin, listeriolisin O (LLO) ve iki ek fosfolipazları 8 aracılık ettiği gösterilmiştir. Sitoplazmada bakteriler (Şekil 1) hücre içindeki aktin filamentler kendilerini itmek için ve hücreden hücreye yayılan konak aktin polimerizasyonu makineleri kullanıyoruz. L. Bütün büyük virulans faktörleri işgali, hücre içi yaşam ve çoğaltmaya katılan monocytogenes, ana virülans transkripsiyon regülatör PrfA. 8-10 aktive edilir. Son on yılda, çeşitli çalışmalar bize tarafından yürütülen ve diğerleri başarıyla konakçı hücrelere 2,11-15 içinde hücre yetiştirilen bakterilerin transcriptome analizi için yöntemler uyguladınız. sorumluluk yönün bakteriyel RNA 1) seçici zenginleştirme ve 2) RNA izolasyonu: İki temel yaklaşım dayanmaktadır konak-RNA bakteriyel RNA ayırmak için kullanılırrential hücre parçalama. Birinci yaklaşım (ticari olarak temin edilebilen kitler kullanılarak, örneğin), memeli RNA moleküllerinin toplam RNA özleri eksiltici hibridizasyon ve bakteriyel transkribe dizileri (SCOTS) 11 seçmeli tutma dayanır. İkinci yaklaşım, bakteriyel hücreler gibi kalır, ev sahibi hücrelerin lize olan bakteri ve konakçı hücreler, ayırıcı liziz dayanır. Bakteriyel hücreler daha sonra, genellikle santrifüjleme ile konakçı hücre lizatından ayrılır ve RNA, standart teknikler kullanılarak ekstre edilir. Bu yaklaşımı kullanarak, ana problem, sağlam bakteri konakçı hücreler çekirdekleri de izole ile birlikte, bu şekilde RNA preparatları de memeli RNA içeren bir. Bu sorunun üstesinden gelmenin bir yolu, bu prosedür genellikle çıkarma sırasında gen tanımlama profili değişikliklerin endişesini zaman alır ancak diferansiyel santrifüj kullanılarak konakçı hücreler çekirdeklerden sağlam bakteri ayırmaktır. Bu yazıda gelişmiş ve hızlı ba sunmakHücre diferansiyel parçalama yaklaşıma dayanmaktadır cteria RNA ekstraksiyon protokolü. İlk olarak, L. makrofaj hücreleri enfekte monocytogenes soğuk su ile lize edilmiştir. Daha sonra, makrofaj çekirdekler kısa bir santrifüjleme ile ayrılmış ve sağlam bakteri hızlı, RNA bakteriyel nükleik asitlerin sıcak fenol-SDS ekstraksiyonu kullanılarak izole edilir, filtre üzerinde toplanır.

<table><tbody><tr><td>Listeria monocytogenes 10403S</td><td></td><td></td><td>&lt;sup&gt;20&lt;/sup&gt;</td></tr><tr><td>Bone marrow derived macrophages prepared from C57B/6 female mice</td><td></td><td></td><td>&lt;sup&gt;21&lt;/sup&gt;</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, RNAse free</td><td>Thermo Scientific</td><td>10977-015</td><td>DEPC-treated water can be used</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41965039</td><td></td></tr><tr><td>Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360-088</td><td></td></tr><tr><td>&amp;#946;-Mercaptoethanol</td><td>Gibco</td><td>31350010</td><td></td></tr><tr><td>Pen/Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10270106</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline-PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Brain heart infusion (BHI)</td><td>Merckmillipore</td><td>1104930500</td><td></td></tr><tr><td>Phenol saturated pH 4.3</td><td>Fisher</td><td>BP1751I-400</td><td></td></tr><tr><td>Chloroform</td><td>Fisher</td><td>BP1145-1</td><td></td></tr><tr><td>Iso-amyl alcohol</td><td>Sigma-Aldrich</td><td>W205702</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>W302406</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>DNaseI</td><td>Fermentas</td><td>EN0521</td><td></td></tr><tr><td>SDS 10%</td><td>Sigma-Aldrich</td><td>L4522</td><td></td></tr><tr><td>Ethanol absolute</td><td>Merck Millipore</td><td>1070174000</td><td></td></tr><tr><td>37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; forced-air incubator</td><td>Thermo Scientific</td><td>Model 3111</td><td></td></tr><tr><td>Cell scrapers</td><td>Nunc</td><td>179693</td><td></td></tr><tr><td>Kontes glass holder for 45 mm filters</td><td>Fisher</td><td>K953755-0045</td><td></td></tr><tr><td>MF-Millipore filters 45&amp;nbsp;mm, 0.45&amp;nbsp;&amp;micro;m</td><td>Merck Millipore</td><td>HAWP04700</td><td></td></tr><tr><td>SpeedVac system</td><td>Thermo Scientific</td><td>SPD131DDA</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries</td><td>Model G560E</td><td></td></tr><tr><td>NanoDrop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>145 mm cell culture dishes</td><td>Greiner</td><td>639 160</td><td></td></tr><tr><td>1.7 ml tubes, RNase-free</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>30 &amp;deg;C incubator</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>65 &amp;deg;C heat block</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>4 &amp;deg;C table centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Sterile pipettes, 25 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Falcon tubes, 50 ml</td><td>Greiner</td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr></tbody></table>

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.

Model sistem, Şekil 1 &#39;de gösterilen ve L ile enfekte makrofaj hücreleri içerir makrofaj sitozolde çoğaltmak bakterileri monocytogenes. 2 deney düzeni temsil Şekil. Şekil 3 WT L. sırasında virülans genlerinin RT-qPCR analizi tipik sonuçlarını temsil Zengin laboratuar ortamı BHI büyümeye kıyasla makrofajlar büyüme monocytogenes. Sonuçlar L. iki ana has...

Watch this Scientific Journal Video about RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells at JoVE.com

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. ...

rna-purification-from-intracellularly-grown-listeria-monocytogenes

Research

JoVE Journal

Immunology and Infection

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

9.1K Görüntüleme.  Tel Aviv University. 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.

Video: RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Yetişkin Karaciğer Hücrelerinin Karma İlköğretim Kültürler Makrofajlar yalıtın Basit ve Etkin Yöntem

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

İmmünoloji ve Enfeksiyon

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

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

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

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

Ile Enfekte Fare Bakteriyel Yük ve Bağışıklık Yanıtları Ölçüm Listeria monocytogenes</em

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

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

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 ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

Sözlü İletim<em&gt; Listeria monocytogenes</em&gt; Kirlenmiş Gıda Yutma ile Farelerde

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

Bakteriyel Patojen Hücresel Enfeksiyon Soruşturma inlc salgılanmasına Görüntüleme<em&gt; Listeria monocytogenes</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

Fare Makrofagların yılında Doğuştan Bağışıklık Tepkisi Soruşturma RNA girişimi kullanma

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or directly into another cell, Gram-negative bacteria can also form outer membrane vesicles (OMVs). These membrane vesicles can deliver their cargo over long distances, and the cargo is protected from degradation by proteases and nucleases. 
Legionella pneumophila (L. pneumophila) is an intracellular, Gram-negative pathogen that causes a severe form of pneumonia. In humans, it infects alveolar macrophages, where it blocks lysosomal degradation and forms a specialized replication vacuole. Moreover, L. pneumophila produces OMVs under various growth conditions. To understand the role of OMVs in the infection process of human macrophages, we set up a protocol to purify bacterial membrane vesicles from liquid culture. The method is based on differential ultracentrifugation. The enriched OMVs were subsequently analyzed with regard to their protein and lipopolysaccharide (LPS) amount and were then used for the treatment of a human monocytic cell line or murine bone marrow-derived macrophages. The pro-inflammatory responses of those cells were analyzed by enzyme-linked immunosorbent assay. Furthermore, alterations in a subsequent infection were analyzed. To this end, the bacterial replication of L. pneumophila in macrophages was studied by colony-forming unit assays. 
Here, we describe a detailed protocol for the purification of L. pneumophila OMVs from liquid culture by ultracentrifugation and for the downstream analysis of their pro-inflammatory potential on macrophages.

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Here, we describe the purification of Legionella pneumophila (L. pneumophila) outer membrane vesicles (OMVs) from liquid cultures. These purified vesicles are then used for the treatment of macrophages to analyze their pro-inflammatory potential.

Legionella pneumophila Dış Zar veziküller: İzolasyon ve Makrofajlar Onların Pro-inflamatuar Potansiyeli Analizi

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

Bakteriyel Pathogen sonda için hücresel ve Organismic düzeyi ana bilgisayar yanıt için kullanma

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

Keşif Odaklı Konak-Patojen Etkileşimleri için Etiketsiz Nicel Proteomik İş Akışı

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Flow Cytometry Analysis of Intracellular and Emergent Extracellular Listeria monocytogenes In Vitro

Source: Gayle, P. et al., <a href="https://app.jove.com/v/58775">Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses.</a> J. Vis. Exp. (2019)
In this video, we quantify intracellular and extracellular Listeria monocytogenes, an intracellular pathogenic bacteria, following their infection of cultured macrophages in vitro using flow cytometry.

İntrasellüler ve Acil Ekstraselüler Listeria monocytogenes'in In Vitro Flow Sitometri Analizi

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp. (2019)
In this video, we ...

RNA Arıtma Hücre içindeki dan Yetiştirilen

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Yetişkin Karaciğer Hücrelerinin Karma İlköğretim Kültürler Makrofajlar yalıtın Basit ve Etkin Yöntem

Ile Enfekte Fare Bakteriyel Yük ve Bağışıklık Yanıtları Ölçüm Listeria monocytogenes

Patojen Vakuoller saflaştırılması gelen

Sözlü İletim

Bakteriyel Patojen Hücresel Enfeksiyon Soruşturma inlc salgılanmasına Görüntüleme

Fare Makrofagların yılında Doğuştan Bağışıklık Tepkisi Soruşturma RNA girişimi kullanma

Legionella pneumophila Dış Zar veziküller: İzolasyon ve Makrofajlar Onların Pro-inflamatuar Potansiyeli Analizi

Bakteriyel Pathogen sonda için hücresel ve Organismic düzeyi ana bilgisayar yanıt için kullanma

Keşif Odaklı Konak-Patojen Etkileşimleri için Etiketsiz Nicel Proteomik İş Akışı

İntrasellüler ve Acil Ekstraselüler Listeria monocytogenes'in In Vitro Flow Sitometri Analizi