Bu çalışma NIH hibe MDP için AI56168, AI71142, AI87541 ve AI100631 tarafından desteklenmiştir. Maurizio Del Poeta Enfeksiyon Hastalıkları Burroughs Wellcome Araştırmacı olduğunu.

MβCD ile J774A.1 Hücreleri 1. Kolesterol tükenmesi <ol><li> Steril bir biyo-güvenlik kabini, tohum 10 5 J774A.1 makrofaj-benzeri hücreler, göz başına,% 10 fetal sığır serumu (FBS) ile takviye edilmiş Dulbecco Modified Eagle Medium (DMEM) içinde 200 ul% 1, 96-iyi hücre kültür plakası üzerine penisilin / streptomisin (P / S). 37 ° C&#39;de inkübe edilir ve% 5 CO2 O / N. </li><li> Hücre mono tabakasının ortamı çıkarın ve süzüldü otoklave edilmiş 1 x fosfat tamponlu tuzlu su (PBS) ile iki kez yıkama hücreleri. </li><li> Bir kontrol olarak, istenen konsantrasyonda (10, PBS içinde mM ya da 30 nM) ya da 1x PBS&#39;den MβCD çözeltisi 200 ul ilave edin ve çalkalanarak 37 ° C&#39;de 30 dakika inkübe edilir. Hemen prosedürü izleyerek piyasada mevcut kiti ile kantitatif analiz için oda sıcaklığında yüzer ve rezerv çıkarın. </li><li> 1x PBS veya serum ücretsiz DMEM ile hücreler 2-3 kez yıkayın ve pipetti tarafından enfeksiyon veya lize hücreleri ile devamince tabaka kromatografisi ile ya da bir kit ile analiz için deiyonize H2O ile 2-3 kez ng. NOT: Bir piyasada mevcut kolesterol ölçümü kiti kullanılabilir kolesterol tükenmesi sonrasında. Ayrıntılar için malzeme bölümüne bakın. Yazılı olarak üreticinin talimatlarını izleyin. </li></ol> İnce Tabaka Kromatografisi ile kolesterol içeriğinin 2. Gözlem (TLC), <ol><li> Petrol eter çözeltisi ile, iki kez aseton ile ve bir kez bir TLC tankı yıkayın: dietil eter: asetik asit (65: 30: 1 hacım bazında). (Hacme göre 1 65: 30) solüsyonu ve / N O bırakmak asetik asit: dietil eter: petrol eteri ile tankı Doymuş. Not: Organik çözücü, her zaman Buhar, önlemek için bir davlumbaz altında kullanılmalıdır. Eldiven ve laboratuvar önlüğü her zaman giyilmelidir. Asetik asit güçlü bir asittir ve dikkatli bir şekilde kullanılmalıdır. </li><li> 6-çukurlu ce üzerinde oyuk başına steril biyo-güvenlik kabini, tohum 10 6 J774A.1 makrofaj benzeri hücre% 10 FBS ve% 1 P / S ilave edilmiş, sıcak DMEM, 5 ml lik bir hacim içinde ll kültür plakası. % 5, 37 ° C&#39;de inkübe CO2 göre O / N. </li><li> 1.4 büyük kuyu boyutu hesaba uygulanan 200 ul için 1 ml ikame - adımları 1.2 izleyerek kolesterol makrofajlar tüketmek. </li><li> Her bir oyuğa Tripsin-EDTA, 500 ul ekle 37 ° C &#39;de 3 dakika süre ile inkübe edilir ve yavaşça bir hücre kazıyıcı ile hücrelerin kazıyın. </li><li> Bir mikrofüj tüpü içine transferi ve% 10 FBS ve% 1 P / S ilave edilmiş, sıcak DMEM ek bir 500 ul ilave edin. </li><li> 300 x g&#39;de 5 dakika boyunca hücreleri Spin ve süpernatant kaldırmak. </li><li> Hücre pelet% 10 FBS ve% 1 P / S ile takviye sıcak DMEM ek 500 ul ekleyin ve yukarı ve aşağı pipetleme dikkatlice tekrar süspansiyon. </li><li> Hücrelerin 10 ul çıkarın ve bir hemasitometre hücreleri saymak. Her bir örnek hücre konsantrasyonları normalize ve cam tüplere hücrelerin eşit sayısı ekleyin. NOT: Bu aşamada, bir mayısBir daha konsantre son lipid özü elde etmek için aynı tedavi grupları hücreleri birleştirmek için seçin. </li><li> Santrifüj hücreleri ortam oda sıcaklığında 5 dakika boyunca 300 x g&#39;de çıkarın. </li><li> Metanol ve vorteks 2 ml ekleyin. Daha sonra, kloroform ve girdap 1 ml. Monofazik emin çözüm yapmak için faz durumunu kontrol edin. Not: tüpler 4 ° de CO / N saklanabilir. </li><li> Oda sıcaklığında 10 dakika boyunca 1700 x g&#39;de santrifüj ve yeni bir tüpe süpernatantı aktarın </li><li> DH 2 O, 1 ml, ardından kloroform ilave edilmiş, 1 ml ilave edilir Iki kez 30 saniye vorteksleyin. Oda sıcaklığında 5 dakika boyunca 1700 x g&#39;de santrifüjleyin. </li><li> Hassas bir denge üzerinde bir cam tüp tartılır ve ağırlığı bilinen cam tüp içine alt faz aktarmak için bir cam Pasteur pipet kullanın. </li><li> Kuru (yaklaşık 2 saat) kadar bir santrifüj buharlaştırıcı içinde lipitleri kurutun. Kurutuldu lipidler ile tüp tartılır ve kuru lipit ağırlığı hesaplamak. Not: Kuru lipid TLC gerçekleştirmek için hazır olana kadar, -20 ° C de muhafaza edilebilir. </li><li> (- 50 ul genelde 20) ve bir silis TLC plakası üzerindeki seyreltilmiş lipid yükü 20 ul lipid konsantrasyonunun normalleştirmek için yeterli kloroform içinde tutularak kurutulmuş lipitlerin seyreltin. Yük 20, bir standart olarak kloroform 20 ul seyreltilmiş kolesterol ug. </li><li> Doymuş TLC tankına kurutulmuş TLC plakası ekleyin ve plaka kenar önce 1 cm kadar göç solvent sağlar. Tanktan TLC plakasını çıkarın ve yaklaşık 5 dakika kurumasını bekleyin. </li><li> Göçü kontrol etmek için bir iyodin buhar tankında yerleştirerek lipit görselleştirmek. Çıkarın ve lekeler yaklaşık 10 solmaya izin - kaputun altında 15 dakika. NOT: İyot bir inhalasyon tehlikesi. Her bir davlumbaz altında kullanın. </li><li> Deiyonize H2O 60 ml, sülfürik asit, 4 ml ve mangan klorür 630 mg 60 ml metanol un birleştirilmesi ile nötral bir lipid boyama için bir çözelti hazırlayın. </li><li> Dikkatle ve yavaş yavaş bir tepsiye nötral lipid boyama çözeltisi içine TLC plakası daldırma ve silis l kapalı sloughing olmadan kaldırmakAyer. Not: nötral lipid boyama çözeltisi bir kaç kez tekrar edilebilir ve bu tepsinin alınabilir ve daha sonraki kullanım için bir şişe içinde geri bırakılır. </li><li> Tüm kabarcıklar kaybolana kadar plaka oda sıcaklığında kaputun altında kurumaya bırakın. Istenilen renkte bir ısıtma bloğu 160 ° C&#39;ye set ve karakter üzerinde TLC plakası ısıtın. NOT: Böyle Vizyon İşleri LS gibi bir dansitometri programı lipit örnekleri karşılaştırmak için kömürleşmiş bantları ölçmek için kullanılabilir. </li></ol> C ile Makrofagların 3. Enfeksiyon neoformans (H99) <ol><li> Steril bir biyo-güvenlik kabininde, DMEM 200 ul, 96 gözlü bir hücre kültürü plakasında tane 10 5 makrofaj-benzeri hücreler,% 10 FBS ve% 1 P / S ile takviye edilmiştir. 37 ° C,% 5 CO2,% 5 CO2 göre O / N inkübe edin. Not: Enfeksiyon cam yapılabilir kolay görüntüleme için konfokal yemekler dipli; Tüm tutarlar aynı kalır. </li><li> C&#39;lik bir kültür büyütün neoformans (H99)Çakılmış plaka elde edilen bir koloni ile YNB 10 ml aşılama ve çalkalanarak 30 ° C&#39;de g / Ç N inkübe edilerek. </li><li> Yıkayın ve C sayısı neoformans (H99) hücreleri. <ol><li> Santrifüj C. 4 ° C&#39;de 10 dakika boyunca 1,700 x g hızında neoformans O / N kültürü. </li><li> Ortamı çıkarın ve atın. 1x PBS, 5 ml hücreleri yıkayın. 4 ° C&#39;de 10 dakika boyunca 1700 x g&#39;de santrifüjleyin. </li><li> PBS çıkarın ve süzüldü 1x 5 ml PBS ile yıkayın. 4 ° C&#39;de 10 dakika boyunca 1700 x g&#39;de santrifüjleyin. Bu adımı 2 kez daha tekrarlayın. </li><li> PBS çıkarın ve 1 x PBS, 5 ml olarak yeniden süspanse edin. </li><li> Yıkandı, kültür: 500 oranında seyreltilmiş PBS&#39;de bir seri seyreltme, 1 elde etmek üzere olun. <ol><li> 1:10 seyreltme elde etmek için 1x PBS 900 ul orijinal numune 100 ul ekle. </li><li> 100 seyreltme: 1 elde etmek için 1x PBS 900 ul 1:10 seyreltilmiş numune, 100 ul ekle. </li><li> 500 diluti bir 1: elde etmek için 1x PBS 800 ul seyreltilmiş örnek, 100: 1 200 ul ekleüzerinde </li></ol></li><li> 1 10 ul alın: 500 seyreltme ve hücrelerin sayısını hesaplamak için hemasitometre saymak. </li></ol></li><li> C makrofajlar aktive ve opsonizasyon için çalışma çözeltisi hazırlayın neoformans. <ol><li> 990 ul 10 ul ekleyerek stok çözümleri LPS ve IFNy 100x seyreltin. Not: LPS IFNy fagositik alımını güçlendirmek üzere kullanılır ancak fagositosis için gerekli değildir. Makrofaj fungisit aktivite, bir ilgi ise, enfeksiyondan önce çalkalama ile 37 ° C&#39;de etkinleştirme O / N gerçekleştirin. </li><li> Numune başına seyreltilmiş LPS 7.5 ul, seyreltilmiş IFNy 1.25 ul, GXM antikor 1.25 ul ve C hacmini birleştirmek 1.25 x 10 5 hücre verir neoformans kültürü. % 10 FBS ve% 1 P / S ile desteklenmiş DMEM ile numune sayısı ile çarpılır 250 ul kadar hacmi getirin. </li><li> Vorteks ve çalkalanarak 37 ° C&#39;de 20 dakika boyunca çözelti inkübe edin. NOT:Opsonizasyon adım ile eş zamanlı olarak yapılabilir - Kolesterol tükenmesi (1.4 1.2 adımları). Opsonize edilmiş hücreler optimum adım 3.4.3 inkübasyon aşağıdaki 20 dakika daha uzun kullanılması gerektiğini gibi, çalışma çözümü birleştirerek önce makrofajlar tedavi emin olun. </li></ol></li><li> Makrofajları enfekte <ol><li> Yıkama makrofajlar iki kez serum serbest DMEM ile ve opsonize C 200 ul ekleyin neoformans her iyi çözüm çalışıyor. </li><li> 37 ° C&#39;de 2 saat süreyle inkübe edin. </li></ol></li><li> Düzeltme ve Leke Hücreler <ol><li> Ortamı çıkarın ve DMEM ile hücreler 2 kez yıkayın. </li><li> Hava, 10 dakika boyunca, hücre tekli tabakası kuru ve hücreleri düzeltmek için buz gibi soğuk metanol 200 ul ilave edin. </li><li> 15 dakika oda sıcaklığında inkübe edin ve kalan metanolü uzaklaştırmak. </li><li> 10x Giemsa 200 ul ilave edin ve oda sıcaklığında 5 dakika inkübe edilir. </li><li> Kapalı kapaklı iyonu giderilmiş su ve kuru O / N ile 3 kez 2 - yıkayın. NOT: Görüntüleme haftada followi ertesi gün yapılması veya yukarı olabilirng boyama. </li></ol></li><li> Görselleştirmek ve Kont <ol><li> Bir mikroskop (oyuk başına 150 sayısı, aynı tedavi 2 varsa) veri alanına 300 hücrelerin sayısı ve saran, Cryptococcus hücrelerinin enfekte olmuş makrofaj sayısı ve sayısı not edin. NOT: durumun başına veri noktası kullanım başına 2 tabak örnekleme bile emin plaka başına 3 örtüşmeyen alanları seçmek ve alan başına 50 hücreleri saymak için. </li><li> C ortalama sayısına göre enfekte makrofajlar yüzdesini çarpılarak fagositik indeksi hesaplamak makrofaj başına neoformans. 1x PBS yüzde kontrolünü ele gibi değerleri ifade ederek fagositik indeksi Normale. Birkaç denemeden sonra ortalama değeri ve fagositik indeksi eğilimleri belirlemek için ortalama standart sapmayı hesaplamak. Önemini belirlemek için Student t-testi kullanın. </li><li> 1,000X veya 400X büyütmede hücrelerin mikrograflanm alın. </li></ol></li></ol> 4. Tripan Mavisi Deneyi <ol><li> Steril bir biyo-güvenlik kabini, tohum 10 6, makrofaj-benzeri hücreler, göz başına bir hacimde bir 6-yuvalı hücre kültürü plakasının üzerindeki 5% 10 fetal büyükbaş hayvan serumu ve% 1 penisilin / streptomisin ile takviye edilmiş Dulbecco minimal esas ortamı (DMEM) içinde mi . % 5, 37 ° C&#39;de inkübe CO2 göre O / N. </li><li> Daha büyük kuyu boyutu hesaba uygulanan 200 ul için 2 ml ikame 1.4 - 1.2 adımları takip ederek kolesterol makrofajlar tüketmek. NOT: 1x PBS yanı sıra herhangi bir tedavi öncesinde kazınarak bir kumanda ile tedavi edilen bir kontrolü de emin olun. </li><li> Iyi her 1x PBS 500 ul ekleyin ve hafifçe bir hücre kazıyıcı ile hücreler kazıyın. Bir mikrofuge&#39;de tüp içine aktarın ve hafifçe yukarı ve aşağı pipetleme hücreleri askıya. </li><li> % 4 Tripan mavi 1 ul hücre ve leke, 10 ul çıkarın. </li><li> Bir hemasitometre hücre sayımı ve aşağıdaki denklem kullanılarak sağ kalabilirliğinin hesaplanması:% canlılığı = [1 - (Mavi hücre / toplam hücre)]Tedavi edilmeyen oldu kontrolü x 100 Normale değerleri. Birkaç denemeden sonra ortalama değeri ve canlılığı eğilimleri belirlemek için standart sapmayı hesaplar. Önemini belirlemek için Student t-testi kullanın. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+To+The+Cryptococcus+Neoformans+Capsular+Glucuronoxylomannan+Are+Ubiquitous.">Antibodies To The Cryptococcus Neoformans Capsular Glucuronoxylomannan Are Ubiquitous.</a> In Serum From HIV+ And HIV- Individuals. Clinical And Experimental Immunology. 99 (3), 425-432 (1995).</li><li>Tripathi, K., Mor, V., Bairwa, N. K., Del Poeta, M., Mohanty, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyurea+Treatment+Inhibits+Proliferation+Of+Cryptococcus+Neoformans+In+Mice.">Hydroxyurea Treatment Inhibits Proliferation Of Cryptococcus Neoformans In Mice.</a> Frontiers In Microbiology. 3, 187 (2012).</li><li>Chaka, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Analysis+Of+Phagocytosis+And+Killing+Of+Cryptococcus+Neoformans+By+Human+Peripheral+Blood+Mononuclear+Cells+By+Flow+Cytometry.">Quantitative Analysis Of Phagocytosis And Killing Of Cryptococcus Neoformans By Human Peripheral Blood Mononuclear Cells By Flow Cytometry.</a> Clin. Diagn. Lab. Immunol. 2 (6), 753-759 (1995).</li></ol>

The authors have nothing to disclose.

Bu protokol ile çalışırken bu C memeli hücreleri kaplama ve opsonizasyon zaman doğru hücre sayılarını elde etmek önemlidir neoformans hücreleri. Bu çalışmalar arasındaki varyasyon en aza indirir ve doğru 1 sağlar: çalışma boyunca efektör oranı 1 hedef. Ayrıca prosedürler arasında oda sıcaklığında istirahat opsonize maya hücreleri ya da muamele edilmiş makrofaj hücreleri önlemek için kolesterol azaltımı ve enfeksiyon zamanlamasını koordine edecek tarzda önemlidir....

Fagositoz hücre dışı kişiler konakçı hücreler tarafından olan bir süreçtir. Bu patojenlere karşı savunmak için bağışıklık sisteminin cephanelik önemli bir silahtır, ama süreç genellikle içselleştirme ve vücutta 1 yayılan sağlamak için patojenler tarafından yıkılabilir olabilir. Fagositoz konukçu hücrenin hücre iskeletinin yeniden düzenlemeler yoluyla bağlanması ve yutulma neden olan bazı sinyal olayları aracılık eder. &quot;Profesyonel&quot; fagositler bağlanması ve patojen yutmak ve phagosome 2 oluşturan lamellipodia oluşması için sinyal istilacı patojenin yüzeyinde opsonins ayırt eden ve bağlanan edebiliyoruz. Sözde &#39;profesyonel&#39; fagositler arasında makrofajlar. Makrofajlar çoğu, arayan ve hastalığa neden olan ajanlar ortadan kaldırarak, hasarlı dokuların onarımı ve inflamasyon aracılık dahil koruyucu fonksiyonlarını yürütmek son derece uzmanlaşmış hücrelerdirfagositoz 1,2 sürecinde bu. Cryptococcus neoformans cryptococcosis olarak bilinen ciddi bir hastalığa neden patojenik maya türüdür. Cryptococcus sporlar ev sahibi tarafından inhale ve genellikle asemptomatik bir akciğer enfeksiyonu neden vardır. Bu poz son derece yaygın olduğu düşünülmektedir; Ankete tüm bu kriptokok polisakkarit glucuronoxylomannan ve diğer çalışmalara antikor olduğunu Kliniği Bronx-Lübnan Hastanesi Merkezi&#39;nde bulunan Çocuk Enfeksiyon Hastalıkları 61 çocuklu bir örnek, hem insan immün yetmezlik virüsü (HIV) enfekte olmayan ve enfekte yetişkinlerin 3 prevalansı göstermiştir 4. Alveoler makrofajlar başarıyla net patojen akciğer enfeksiyonuna ve çoğu durumda yanıtın ilk satırı vardır. Ancak, bağışıklığı baskılanmış kişilerde (örneğin, HIV ve AIDS hastaları) maya makrofajlar içinde hayatta yapabiliyor. Bunlardadavalar, makrofajlar patojenin çoğalması için bir niş olarak hizmet verebilir ve hastalık ölümcül hale 5 santral sinir sistemi (MSS) onun yayılmasını kolaylaştırabilir - 8. 11 - Bu makrofajlar hatta &quot;Truva atı&quot; modeli 3,9 yoluyla kan beyin bariyerini geçmeye maya yardımcı meninkslerde doğrudan maya teslim düşünülmektedir. Böylece, bu fagositoz sürecini ve özellikle kriptokoksik enfeksiyonlarda, bunu etkileyen faktörleri anlamak önemlidir. 15 - Diğer patojen sistemlerinde Önceki çalışma fagositozu 12 oynamak için önemli bir role sahip olduğu kolesterol oluşturduğu kolesterol ve lipid sallar işaret. Kolesterol, memeli hücrelerde en fazla lipit türdür ve 25 ihtiva eder - memeli hücre zarı 16, 50%. Bu BIOP modüle bir rol oynadığı tespit edilmiştirrijiditelerini 17 değiştirerek zarların hysical özellikleri. Kolesterol ve sfingolipitler birlikte lipid sallarının olarak bilinen membran lipit mikro bölgelerini oluştururlar. 18 - lipid sallarının kaveolanın oluşumu, hem de 16 sinyal belirli tipleri için, izole edilmiş bir etki sağlayan ilgili olduğu tespit edilmiştir. Nedeniyle, küçük boyutları nedeniyle, in vivo olarak, lipid sallar çalışmak zordur. Lipid sallar rolünü incelemek için yararlı bir yolu, onların bileşenleri değiştirmek için. Metil-β-siklodekstrin (MβCD) memeli zarlarda kolesterol tüketen bulunmuştur ve yaygın olarak lipid sallarının 18 rolünü araştırmak için kullanılan bir bileşimdir. Bu protokol, konakçı hücre zarlarından kolesterol tüketen ve C fagosite konakçı hücrelerin yeteneği üzerindeki tükenmesi etkisinin belirlenmesi için bir yöntem sunmak in vitro neoformans. Bu prosedür, hücre kültürü techniq kullanırenfeksiyonu için bir model olarak hücre hattı (J774A.1) gibi ölümsüzleştirilmiş makrofaj üzerinde daah. Kolesterol tükenmesi sterol büyüklüğüne özel bir hidrofobik bir çekirdeğe sahiptir ve kolesterol zar 19 dışarı çekmek için bir havza olarak etki edebilir MβCD, maruz bırakılarak gerçekleştirilmiştir. Kolesterol tükenmesi kantitatif olarak ticari olarak temin edilebilen bir kit kullanılarak ve niteliksel olarak ince tabaka kromatografisi (TLC), daha sonra da 20 modifiye edilmiş bir Bligh-Dyer lipid ekstraksiyonu kullanılarak ölçülmüştür. . 23 - fagositoz makrofajları aktive edilmesi için, interferon-γ ve lipopolisakkarit bir kokteyl ile karıştırılır opsonize bir maya kültürünün hücre hattı enfekte ölçülmüştür Cryptococcus bir glucuronoxylomannan (GXM) antikor ya 21 ile opsonize edilmiştir. Boyama ve mikroskopi deneyleri fagositoz derecesini değerlendirmek için hücrelerin görselleştirme ve fagositik endeksinin hesaplanmasında izin verdi. Bu protokol d birlikte ele alındığındafizyolojik bir süreç ile, lipid bileşimin değiştirilmesini entegre temel bir metodu escribes.

<table border="0" cellpadding="0" cellspacing="0" width="622">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:120px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Class II type A2 Biosafety Cabinet</td>
			<td style="width:120px;">Labconco</td>
			<td style="width:119px;">3460009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">J774A.1 cell line</td>
			<td style="width:120px;">ATCC</td>
			<td style="width:119px;">TIB-67</td>
			<td style="width:167px;">Arrives Frozen. See ATCC instructions for culturing.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">11995-065</td>
			<td style="width:167px;">Store at 4 &#176;C and warm to 37 &#176;C prior to use</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">HI Fetal Bovine Serum Performance Plus</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">10082-147</td>
			<td style="width:167px;">Keep frozen at -20 &#176;C and thaw before adding to DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:167px;">Used to suplement DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isotemp Cell culture incubator</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # 3530</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well culture dish</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3595</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10x Phosphate Buffered Saline</td>
			<td style="width:120px;">Fisher Scientific BioReagents</td>
			<td style="width:119px;">BP3994</td>
			<td style="width:167px;">Dilute to 1x and filter or autoclave prior to use.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Methyl-&#946;-Cyclodextrin</td>
			<td style="width:120px;">Sigma Life Science</td>
			<td style="width:119px;">C4555-10G</td>
			<td style="width:167px;">Dissolve in 1x PBS to make solutions of 10mM and 30mM concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Orbital Shaker</td>
			<td style="width:120px;">Labline</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Amplex Red Cholesterol Assay Kit</td>
			<td style="width:120px;">Life Technologies Molecular Probes</td>
			<td style="width:119px;">A12216</td>
			<td style="width:167px;">All reagents for Cholesterol Assay are contained within the kit. Follow Manufacturer instructions.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96-Well Black Assay plate</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3603</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FilterMax microplate reader</td>
			<td style="width:120px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model F5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TLC Chamber</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z126195-1EA</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">650498-4L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">34860-2L-R</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TLC Paper</td>
			<td style="width:120px;">Whatman</td>
			<td style="width:119px;">3030917</td>
			<td style="width:167px;">Cut down to size needed for TLC tank</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fume Hood</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Any fume hood that complies with AIHA/ANSI Standards&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">6-Well Plate</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3506</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA</td>
			<td style="width:120px;">Gibco</td>
			<td style="width:119px;">25300-054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Scraper</td>
			<td style="width:120px;">Corning Inc. Costar</td>
			<td style="width:119px;">3010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hemocytometer&#160;</td>
			<td style="width:120px;">Hausser Scientific</td>
			<td style="width:119px;">1490</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:120px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model Alegra x-30R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Votex Mixer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;">12-812</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Balance</td>
			<td style="width:120px;">Mettler Toledo</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # MS104S Meaures down to .1 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Pasteur Pipette</td>
			<td style="width:120px;">Fisherbrand</td>
			<td style="width:119px;">13-678-20A</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cholesterol</td>
			<td style="width:120px;">Avanti Polar Lipids</td>
			<td style="width:119px;">700000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SpeedVac Concentrator</td>
			<td style="width:120px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Model # SPD2010&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petroleum Ether</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:119px;">E139-1&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl Ether</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">309966&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetic Acid</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">320099</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TLC Silica Gel 60 with concentrating zone</td>
			<td style="width:120px;">Analytical Chromatograhy Millipore</td>
			<td style="width:119px;">1.11845.0001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Iodine Chips</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">376558-50G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sulfuric Acid</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">320501&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Manganese Chloride</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:119px;">244589</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">UVP EC3 Imaging System</td>
			<td style="width:120px;">Ultra-Violet Products Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Use the Vision Works LS software for densitometry analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Bottom Confocal Dish</td>
			<td style="width:120px;">MatTek</td>
			<td style="width:119px;">P35G-1.5-10C</td>
			<td>www.glassbottomdishes.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cryptococcus neoformans (H99)</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Obtained from Duke University Medical Center</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">YNB</td>
			<td style="width:120px;">BD</td>
			<td style="width:119px;">239210</td>
			<td style="width:167px;">See manufacturer for preparation instructions. Use a Glucose concentration of 20 g/L.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Lipopolysaccharide</td>
			<td style="width:120px;">Sigma</td>
			<td style="width:119px;">L4391-1MG</td>
			<td style="width:167px;">Dissolve in 1x PBS to make 1mg/mL stock. Store at -20 &#176;C.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Interferon gamma</td>
			<td style="width:120px;">Sigma</td>
			<td style="width:119px;">I4777</td>
			<td style="width:167px;">Dissolve in 1x PBS to make .1 mg/mL stock solution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Glucuronoxylomannan antibody (anti-GXM)</td>
			<td style="width:120px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Gift from Arturo Casadevall&#39;s Lab concentration is 1.98 mg/mL</td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:216px;">Giemsa</td>
			<td style="width:120px;">MP Biomedicals</td>
			<td style="width:119px;">194591</td>
			<td style="width:167px;">Dissolve .8 g of Giemsa in 25 mL of Glycerol and heat to 60 &#176;C for 1 hour. Add 25 mL of methanol to the solution and allow to age at room temperature for at least 1 month.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Microscope</td>
			<td style="width:120px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Observer.D1 microscope with AxioCam MRm for taking images</td>
		</tr>
	</tbody>
</table>

macrophage cholesterol depletion and its effect on the phagocytosis of cryptococcus neoformans

Kriptokokkozis cinsi, Cryptococcus patojenik mantarların neden olduğu bir yaşamı tehdit edici hastalıktır. Enfeksiyon akciğerin derinlerine içinde replike olan sporların, teneffüs sırasında görülür. Makrofajlar tarafından Cryptococcus fagositozu hastalığı, ölümcül meningoensefalit neden merkezi sinir sistemi içine yayabilen yollarından biridir. Bu nedenle, Cryptococcus ve makrofajlar arasındaki ilişkinin incelenmesi enfeksiyonun ilerlemesinin anlamak önemlidir. Bu çalışmada C. makrofaj enfektivitesini incelemek için bir adım-adım protokolü açıklanır in vitro neoformans. Bu protokolü kullanarak, konak-patojen etkileşimleri ev sahibi steroller rolü incelenmiştir. Metil farklı konsantrasyonları - siklodekstrin (MCD), sıçangil sarkomu retikulum makrofaj benzeri hücre hattı J774A.1 kolesterol tüketmek için kullanılmıştır. Kolesterol tükenmesi doğrulandı ve ticari availabl kullanarak hem kantifıyeE kolesterol ölçümü kiti ve ince tabaka kromatografisi. Kolesterol tükenmiş hücreleri lipopolisakkarittir (LPS) ve interferon gamma (IFNy) ile aktive edilir ve 1 arasında bir efektör-hedef oranında antikor opsonize Cryptococcus neoformans, vahşi tip H99 hücreleri ile enfekte edilmiştir: 1 arasındadır. Enfekte hücreler, C ile inkübasyon 2 saat sonra izlenmiştir neoformans ve fagositik indeksi hesaplandı. Kolesterol tükenmesi fagositik indeksinde önemli bir azalma ile sonuçlandı. sunulan protokoller laboratuar ortamında enfeksiyon sürecinin başlatılmasını taklit ve enfekte ev sahibi lipit kompozisyonu rolünü incelemek için uygun bir yöntem sunuyoruz.

Kolesterol tükenmesi 1x PBS kontrol ile karşılaştırıldığında Amplex Red Kolesterol Test kitinde, üretici talimatlarına göre protokol aşama 1.3 ayrılmış yüzer analizi MβCD ile muamele edilen numune kolesterolün yüksek bir konsantrasyonu elde edilir. Hücre tipine ve MβCD konsantrasyonu, kullanılan kolesterol azaltımı bağlı olarak değişebilir. 10 mM MβCD ile muamele J774 için, yaklaşık% 50 oranında bir azalması görülmektedir. Boşaltma 1.4 adım (Şekil...

Watch this Scientific Journal Video about Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans at JoVE.com

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

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

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

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

macrophage-cholesterol-depletion-its-effect-on-phagocytosis

Research

JoVE Journal

Immunology and Infection

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

12.6K Görüntüleme.  Stony Brook University. In this article, a protocol for infection of macrophages with Cryptococcus neoformans is described. Also, a method for sterol depletion from the macrophages is explained. These protocols provide a guide to study fungal infections in vitro and examine the role of sterols in such infections. 

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

Depletion of Specific Cell Populations by Complement Depletion

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

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

Kompleman tükenmesi Özel Hücre Populasyonlarının tükenmesi

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

İmmünoloji ve Enfeksiyon

Depletion and Reconstitution of Macrophages in Mice

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

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

Farelerde Makrofajlar tükenmesi ve Sulandırma

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

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

Recently there were several publications on the bactericidal effect of visible light, most of them claiming that blue part of the spectrum (400 nm-500 nm) is responsible for killing various pathogens1-5. The phototoxic effect of blue light was suggested to be a result of light-induced reactive oxygen species (ROS) formation by endogenous bacterial photosensitizers which mostly absorb light in the blue region4,6,7. There are also reports of biocidal effect of red and near infra red8 as well as green light9.
In the present study, we developed a method that allowed us to characterize the effect of high power green (wavelength of 532 nm) continuous (CW) and pulsed Q-switched (Q-S) light on Pseudomonas aeruginosa. Using this method we also studied the effect of green light combined with antibiotic treatment (gentamycin) on the bacteria viability. P. aeruginosa is a common noscomial opportunistic pathogen causing various diseases. The strain is fairly resistant to various antibiotics and contains many predicted AcrB/Mex-type RND multidrug efflux systems10.
The method utilized free-living stationary phase Gram-negative bacteria (P. aeruginosa strain PAO1), grown in Luria Broth (LB) medium exposed to Q-switched and/or CW lasers with and without the addition of the antibiotic gentamycin. Cell viability was determined at different time points. The obtained results showed that laser treatment alone did not reduce cell viability compared to untreated control and that gentamycin treatment alone only resulted in a 0.5 log reduction in the viable count for P. aeruginosa. The combined laser and gentamycin treatment, however, resulted in a synergistic effect and the viability of P. aeruginosa was reduced by 8 log's.
The proposed method can further be implemented via the development of catheter like device capable of injecting an antibiotic solution into the infected organ while simultaneously illuminating the area with light.

The Synergistic Effect of Visible Light and Gentamycin on Pseudomona aeruginosa Microorganisms

We show that a developed biomedical device involving continuous or pulsed visible laser based treatment that is combined with antibiotic treatment (gentamycin), results in a statistically significant synergistic effect leading to a reduction in the viability of P. aeruginosa PAO1, by 8 log's compared to antibiotic treatment alone.

Üzerinde görebilir Işık ve Gentamisin en Sinerjik Etkisi<em&gt; Pseudomona aeruginosa</em&gt; Mikroorganizmalar

Recently there were several publications on the  bactericidal effect of visible light, most of them claiming that blue part of  the spectrum (400 nm-500 ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

Ana-Hücre RNA sentezi üzerinde Viral Enfeksiyon Etkisi Tedbir tıklayın Kimya Kullanımı

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon.

Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

We describe how to visualize macrophage-C. neoformans (Cn) interactions in real time, with specific emphasis on the process of non-lytic exocytosis using digital light microscopy. Using this technique individually infected macrophages can be studied to ascertain various aspects of this phenomenon.

Sigara litik Ekzositoz görselleştirme<em&gt; Cryptococcus neoformans</em&gt; Dijital Işık mikroskopi ile Makrofagların gelen

Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

Makrofaj Fagositoz ve Aktin Polimerizasyon Değerlendirme Yüksek Verimli Florimetrik Tekniği

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide.&#160;Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only&#160;biolistic&#160;delivery provides&#160;an effective means to transform&#160;linear&#160;DNA&#160;that can be integrated into the genome by homologous recombination.&#160;
Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known.&#160;A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into&#160;C. neoformans. We then confirm integration of the ACK-mCherry fusion into the&#160;ACK&#160;locus.

Biolistic Transformation of a Fluorescent Tagged Gene into the Opportunistic Fungal Pathogen Cryptococcus neoformans

Biolistic transformation is a method used to generate stable integration of DNA into the genome of the opportunistic pathogen Cryptococcus neoformans through homologous recombination. We will demonstrate biolistic transformation of a construct, which has the gene encoding acetate kinase fused to the fluorescent tag mCherry into&#160;C. neoformans.

Fırsatçı mantar patojenlere bir Floresan etiketli geninin biyolistik transformasyonu<em&gt; Cryptococcus neoformans</em

The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal ...

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. ...

Assessment of Antibody-based Drugs Effects on Murine Bone Marrow and Peritoneal Macrophage Activation

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages are equally important in turning off inflammatory responses. We have shown that macrophages stimulated with intravenous immunoglobulin (IVIg) can produce high amounts of the anti-inflammatory cytokine, interleukin 10 (IL-10), and low levels of pro-inflammatory cytokines in response to bacterial lipopolysaccharides (LPS). IVIg is a polyvalent antibody, primarily immunoglobulin Gs (IgGs), pooled from the plasma of more than 1,000 blood donors. It is used to supplement antibodies in patients with immune deficiencies or to suppress immune responses in patients with autoimmune or inflammatory conditions. Infliximab, a therapeutic anti-tumor necrosis factor alpha (TNF&#945;) antibody, has also been shown to activate macrophages to produce IL-10 in response to inflammatory stimuli. IVIg and other antibody-based biologics can be tested to determine their effects on macrophage activation. This paper describes methods for derivation, stimulation, and assessment of murine bone marrow macrophages activated by antibodies in vitro and murine peritoneal macrophages activated with antibodies in vivo. Finally, we demonstrate the use of western blotting to determine the contribution of specific cell signaling pathways to anti-inflammatory macrophage activity. These protocols can be used with genetically modified mice, to determine the effect of a specific protein(s) on anti-inflammatory macrophage activation. These techniques can also be used to assess whether specific biologics may act by changing macrophages to an IL-10-producing anti-inflammatory activation state that reduces inflammatory responses in vivo. This can provide information on the role of macrophage activation in the efficacy of biologics during disease models in mice, and provide insight into a potential new mechanism of action in people. Conversely, this may caution against the use of specific antibody-based biologics to treat infectious disease, particularly if macrophages play an important role in host defense against that infection.

Antibody-based drugs have revolutionized treatment for inflammatory diseases. In addition to having direct effects on specific targets, antibodies can activate macrophages to become anti-inflammatory. This protocol describes how anti-inflammatory macrophage activation can be assessed in vitro, using mouse bone marrow macrophages, and in vivo, using peritoneal macrophages.

Fare kemik iliği ve Periton makrofaj harekete geçirmek antikor bazlı ilaçlara etkileri değerlendirilmesi

Macrophages are phagocytic innate immune cells, which initiate immune responses to pathogens and contribute to healing and tissue restitution. Macrophages ...

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size can vary widely between strains, has the ability to grow rapidly when introduced to stressful or low nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. The growth of the C. neoformans capsule is induced during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here we describe one of the standard methods of capsule induction and compare two accepted methods of staining and measuring capsule diameter: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we show how measurement of capsule diameter from India ink-stained samples can be automated using computational image analysis.

Size Matters: Measurement of Capsule Diameter in Cryptococcus neoformans

The polysaccharide capsule is the primary virulence factor in Cryptococcus neoformans, and its size correlates with strain virulence. Capsule diameter measurements are used in phenotypic testing and to gauge therapeutic efficacy. Here a standard method of capsule induction is presented, and two methods of staining and measuring diameter are compared.

Size Matters: Cryptococcus neoformans kapsül çap ölçümü

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic ...