Bu araştırma NIH ödül 5T32AI07506, 5R01AI033774, 5R37AI033142, 5R01AI052733 tarafından kısmen desteklenmiştir.

Tüm hayvan çalışma mevzuatına ve Albert Einstein Tıp Koleji Hayvan Araştırmaları Enstitüsü kurallarına uygun olarak yapıldı. C. 1. Yetiştirilmesi neoformans H99 (serotip A) <ol><li> Bireysel C büyütün Sabouraud agar plakaları üzerinde neoformans (Cn) kolonileri. </li><li> Bir gün önce deney, tek bir koloni seçip Sabouraud suyu 10 ml aşılamak. </li><li> Kültür 250 rpm bir hızda çalkalanarak 37 ° C&#39;de bir gece boyunca büyümeye izin verin. </li></ol> 2. İzolasyon ve C57 Primer Kemik Makrofagların Hazırlık / BL6 Fareler <ol><li> Artık nefes kadar bir CO2 odasına hayvana yerleştirerek fareler öldürülür. İkinci bir önlem olarak, cervically diseksiyonu başlamadan önce, boyun yerinden. % 70 etil alkol ile tüm vücut sterilize. </li><li> Steril DMEM&#39;de hem femur ve tibia ve yer kemikleri çıkarın. </li><li> Aşırı doku ve kas DE kullanarak sökünKemikler tamamen temizlenene kadar licate mendil. </li><li> % 70 etil alkol içeren bir Petri kutusu sadece sağlam kemikleri yerleştirin ve ilgili mikroorganizmaları öldürmek için 3 dakika boyunca inkübe edin. Steril DMEM ile Petri kabındaki kemikleri ve yer çıkarın. </li><li> Dikkatle kemiklerin uç kesti. Boş bir 50 ml konik tüp, buz üzerine yerleştirildi ve her bir kemik içinden bir 25 G iğne kullanılarak soğuk DMEM, 10 ml dikkatli bir hizada fazla kemik tutun. NOT: Her fare 4 kemikleri (2 femur, tibia 2) verecektir. Bu nedenle, bir fare hücre süspansiyonu yaklaşık olarak 40 ml elde edilmelidir. </li><li> 650 x g&#39;de 10 dakika boyunca oda sıcaklığında santrifüje hücre süspansiyonu. </li><li> Bu Santrifüj sırasında hazırlamak ve filtre kemik iliği makrofaj% 20 L929,% 10 fetal dana serumu,% 10 NCTC-109,% 1 Pen-strep,% 1 HEPES,% 1 L-glutamin ile takviye edilmiş DMEM ortamı oluşur besleme sterilize , temel olmayan amino asitler,% 1,% 0.1 2-mersaptoetanol eklenir. </li><li> Besleme ortamı 10 ml ile yeniden süspanse edilen topak. Hücre esas asma geçmekhücre kümeleri bozabilir ve büyük enkaz kaldırmak için 70 mikron hücre süzgecinden iyon. </li><li> Levha (10 plakaların toplam) doku kültürü kullanılmak üzere optimize edilmiştir Petri tabaklarında besleme ortam 10 ml hücre süspansiyonu 1 mi. </li><li> Hücreler% 10 CO2 ile 37 ° C &#39;de yapışmaya izin verin. 7. günde, 3. günde taze besleme ortamı 5 ml tam ortam besleme ve makrofajlar kullanılmaya hazırdır değiştirin. </li><li> Deneyden önceki gün, besleme ortamı kaldırarak levhadan ayırmak ve makrofaj plaka reaktif sıyırma yumuşak bir hücrenin 5ml ekleyin. </li><li> 37 ° C&#39;de 5-10 dakika süreyle inkübe ve hafif pipetle makrofajlar çıkarın. </li><li> 10 dakika boyunca 650 x g&#39;de santrifüj ile pelet hücreleri. </li><li> Medya besleme 1 ml içinde süspanse pelet. 1:20 seyreltme kullanılarak, bir haemositometre üzerine hücre süspansiyonundan 10 ul pipetleme makrofajların konsantrasyonunu hesaplamak. </li><li> Petri kapları 14 mm cam tabanı kullanarak, bir plakaşirketinden 200 ul maksimum kapasitesi iç kuyu üzerine 1 x 10 5 makrofaj konsantrasyonu, bu yüzden, Şekil 4&#39;te gösterildiği gibi, bu birimin aşmaması için dikkatli alır. </li><li> Hücreler 1 saat boyunca 37 ° C inkübatör yemekleri yerleştirerek cam bir Petri tabağına yapışma izin verin. </li><li> 500 U / ml, 1 mg / ml IFNg LPS ile takviye edilmiş ortam besleme 1-2 ml ilave edilir ve hücreler bir gece boyunca inkübe edin. </li></ol> Primer sıçan makrofaj lan ve C. 3. alk kuluçkalama neoformans <ol><li> Deney gününde, 420 x g&#39;de 5 dakika boyunca santrifüj edilerek bir gece kültürü aşılandı ve pelet maya hücrelerinin 1 ml&#39;sini uzaklaştırın. </li><li> Yukarıda belirtilen hızda ve zamanda polisakkarit glucuronoxylomannan (GXM) gibi medya ve hücre dışı kriptokoksik ürünleri kaldırmak için steril PBS ile hücreler 3 kez yıkayın. </li><li> 100 seyreltme: steril 1 ml PBS içinde yeniden süspanse ve hücre topağı, 1 olun. T pipetle 10 ulo bir haemocytometer üzerine seyreltme ve hücreleri saymak. 1 MOI&#39;da maya hücreleri ekleyin: 5&#39;tir. , 1 x 10 5 makrofajlar bulunmaktadır, örneğin, / yuva, Cn 5 x 10 6 / ml &#39;lik bir hücre süspansiyonu yapmak ve 5 x 10 5 Cn toplam bir miktarda bu çözeltiye 100 ul ilave edin. </li><li> 10 ug / ml &#39;lik bir konsantrasyonda mAb 18B7 antikor ile besleme ortam 1 ml hücre süspansiyonu, kriptokokal hesaplanmış hacim. 5 dakika boyunca oda sıcaklığında bir hücre süspansiyonu inkübe edin. </li><li> Camdan besleme medyayı çıkartın Petri dipli çanak ve steril PBS ile 1x yıkayın. Iyi doğrudan opsonize Cn 100 ul ekleyin. </li><li> Hücreler% 10 CO2 ile 37 ° C&#39;de 2 saat inkübasyona izin verin. </li><li> Co-kuluçkasından sonra, makrofajlar Cn içselleþtirdikleri bu mikroskop ile kontrol edin. Içselleştirilmesi kabul edilmesi için, kriptokokal hücreler açıkça makrofaj sınırları içinde görülmelidir. </li><li> Steril PBS ile yıkayın Petri kabı, 3x her türlü extracellula kaldırmak içinr Cn. </li><li> 1-2 mAb 18B7 eklenmeksizin ortam besleme ml ve ayarlamak mikroskop ekleyin. </li></ol> Dijital Işık Mikroskobu kullanarak gerçek zamanlı 4. görselleştirme Non-litik Ekzositoz NOT: CO 2 teslimat ve bir ısıtma ünitesi gerekli içeren ekli bir kuluçka odası ile birlikte bir mikroskop bu deneyler gerçekleştirmek için. <ol><li> Deneyden önce en az 30 dakika 37 ° C&#39;de inkübe Kabin, önceden ısıtın. CO2 birimi Deneyin başlangıcında% 5 okur emin olun. </li><li> Yer cam CO 2 kapaklı kapak ve hiçbir ısı kaçar sağlamak için bütün kapılarını kapatmak, mikroskop platformda Petri dipli. </li><li> Faz kontrast mikroskop ile 10X objektif kullanılarak, enfekte makrofajlar net bir alana odaklanmak. Deney göre, üzerinde çalışılacak gerekir kaç makrofajlar tarafından kullanılan hedefi belirler. </li><li> Ta için mikroskop yazılımı kurmabir görüntüye 24 saatlik bir süre boyunca her 4 dakikada ke. </li><li> 24 saat sonra deney sona ulaştığını olacak ve 361 bir görüntü toplanan toplam olacaktır. 5% sıkıştırmada .jpegs olarak bu görüntüleri aktarın. Resim J yazılımı kullanarak, saniyede 5 kare Visual Stack olarak görüntüleyebilirsiniz. Film yayın veya sunum için inceledi gerekiyorsa, bir avi veya biçim izleyici için en iyi çalıştığını bağlı Mov dosyası ya olarak kaydedin. </li></ol>

<ol>
	<li>Voelz, K., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcal+Interactions+With+the+Host+Immune+System.">Cryptococcal Interactions With the Host Immune System.</a> Eukaryotic cell. 9 (6), 835-846 (2010).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptococcus+interactions+with+macrophages:+evasion+and+manipulation+of+the+phagosome+by+a+fungal+pathogen.">Cryptococcus interactions with macrophages: evasion and manipulation of the phagosome by a fungal pathogen.</a> Cellular microbiology. 15 (3), 403-411 (2012).</li><li>Sabiiti, W., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+infection+by+the+human+fungal+pathogen+Cryptococcus+neoformans.">Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans.</a> Future microbiology. 7 (11), 1297-1313 (2012).</li><li>Bliska, J. B., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pathogenic+bacteria+and+fungi+&#8212;+a+case+of+convergent+evolution.">Intracellular pathogenic bacteria and fungi &#8212; a case of convergent evolution.</a> Nature Reviews Microbiology. 7, 165-171 (2008).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+spread+and+massive+vacuole+formation+after+Cryptococcus+neoformans+infection+of+murine+macrophages.">Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages.</a> BMC immunology. 8 (1), 16 (2007).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cell-to-cell+spread+of+a+pathogenic+yeast.">Direct cell-to-cell spread of a pathogenic yeast.</a> BMC immunology. 8, 15 (2007).</li><li>Alvarez, M., Casadevall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+extrusion+and+host-cell+survival+after+Cryptococcus+neoformans+phagocytosis+by+macrophages.">Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages.</a> Curr Biol. 16 (21), 2161-2165 (2006).</li><li>Ma, H., Croudace, J. E., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expulsion+of+live+pathogenic+yeast+by+macrophages.">Expulsion of live pathogenic yeast by macrophages.</a> Curr Biol. 16 (21), 2156-2160 (2006).</li><li>Johnston, S. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+fungal+pathogen+Cryptococcus+neoformans+escapes+macrophages+by+a+phagosome+emptying+mechanism+that+is+inhibited+by+Arp2/3+complex-mediated+actin+polymerisation.">The human fungal pathogen Cryptococcus neoformans escapes macrophages by a phagosome emptying mechanism that is inhibited by Arp2/3 complex-mediated actin polymerisation.</a> PLoS pathogens. 6 (8), (2010).</li><li>Carnell, M., 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=Actin+polymerization+driven+by+WASH+causes+V-ATPase+retrieval+and+vesicle+neutralization+before+exocytosis.">Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis.</a> The Journal of cell biology. 193 (5), 831-839 (2011).</li><li>Voelz, K., Lammas, D. A., May, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+regulates+the+outcome+of+intracellular+macrophage+parasitism+by+Cryptococcus+neoformans.">Cytokine signaling regulates the outcome of intracellular macrophage parasitism by Cryptococcus neoformans.</a> Infection and immunity. 77 (8), 3450-3457 (2009).</li><li>Bain, M. J., Lewis, E. L., Okai, B., Quinn, J., Gow, A. R. N., Erwig, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-lytic+expulsion/exocytosis+of+Candida+albicans+from+macrophages.">Non-lytic expulsion/exocytosis of Candida albicans from macrophages.</a> Fungal genetics and biology. 49 (9), 677-678 (2012).</li></ol>

Yazarlar hiçbir rakip mali çıkarlarını veya diğer ilgi çatışmaları olmadığını beyan ederim.

Burada, mantar-makrofaj etkileşimleri kaydedildi ve 24 saatlik bir süre boyunca gerçek zamanlı olarak analiz edilebilir bir yöntemi tanımlamaktadır. Bizim laboratuvar olmayan litik ekzositoz zamansal açıdan birçok incelemek için bu protokolü kullandı ve bu süreci çevreleyen morfolojik bileşenleri tespit etmek için gerçek zamanlı mikroskobu kullanmaya devam edecektir. Bu teknik, doğru sonuçlar elde etmek için özel bir dikkat protokol belirli adımlara verilmelidir. Çok...

Kriptokokal hücreleri ve makrofajlar arasında bir mantar enfeksiyonunun safhaları 1-3 belgelenmiştir. Maya hücreleri, makrofajlar tarafından sindirilen sonra etkileşimlerin çeşitli oluşabilir: makrofaj hücre dışı alanı içine mantar yükünü serbest lize olabilir veya hücresel membran 4 sınırları içinde maya hücrelerini tutarak enfeksiyon kontrolü olabilir. Litik olmayan ekzositozu, bir makrofaj çevredeki ortama kriptokokal hücrelerin bazı veya tüm expunged bir işlemdir ya da komşu hücre ve ana ve hem de patojen 5 canlı kalır: Bununla birlikte, birkaç yıl önce, yeni bir sonuç iki grup ile bağımsız bir şekilde tanımlanmıştır -8. Çeşitli çalışmalar bu etkileşimin ardındaki moleküler mekanizmalarını anlamaya çalıştılar ama biz hala bu fenomen neyin tam bir kavrayışa sahip değilsiniz. Yeteneği, gerçek zamanlı görüntü yakalama ve sonradan birden Infect analized makrofajlar bir mantar enfeksiyonu sırasında morfoloji ve makrofajlar hatta stres zamanlaması, mekansal kapasite, değişim ile ilgili olmayan litik eksositoz çevreleyen fiziksel özellikleri hakkında birçok sorulara cevap verir. Hücreler bir kuluçka karşılaştırılabilir bir ortamda muhafaza edilecek izin, biz makrofaj-mantar hücre etkileşimlerinin dinamik doğasını belirti. Araştırmacılar, bu sürecin çeşitli bileşenleri incelemek için bu tekniği kullandık. Başka bir grup litik olmayan ekzositozu protein alanlarının çekirdek YIKAMA 10 silinen oldu hücrelerde bloke olduğunu göstermek için bu yöntemi kullanan Bu işlemde fagozomun rolü, floresan lekelenmesi ve aktin bloke edici maddeleri kullanılarak açık 9 yapılmıştır. Sitokin sinyallerinde etkisi de gerçek zamanlı mikroskobu 11 kullanılarak tespit edilmiştir. Bu işlem, C bunlarla sınırlı değildir bunun gibi neoformans, Candida albicans gözlenmiştir anothemakrofajlar 12 bulaştırmak için yeteneğine sahiptir mantar patojen r. Bu bulgular bu yöntem hakkında o kadar az şey biliyoruz bir alana bir bilgi zenginliği sağlayabilir nasıl örnekleridir.

<table border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sabouraud Dextrose Agar</td>
			<td style="width:183px;">BD</td>
			<td style="width:227px;">DF0109-17-1</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sabouraud Dextrose Broth</td>
			<td>BD</td>
			<td>DF0382-17-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dubelcco&#39;s Modified Eagle&#39;s Medium(DMEM)</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Calf Serum ( FCS)&#160;</td>
			<td>Atlanta Biologicals</td>
			<td>S12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NCTC 109</td>
			<td>Invitrogen</td>
			<td>21340-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES Buffer</td>
			<td>Corning Cellgro</td>
			<td>25-060-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-essential Amino Acids</td>
			<td>Corning Cellgro</td>
			<td>25-025-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3003 Tissue Culture Petri Dishes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>08772E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellStripper</td>
			<td>Corning Cellgro</td>
			<td>25-056-Cl</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mat-tek Glass Bottom Culture Dishes</td>
			<td>MatTek</td>
			<td>P35GC-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LPS</td>
			<td>Sigma</td>
			<td>L3137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-g</td>
			<td>Roche</td>
			<td>NC 9222016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mAb 18B7</td>
			<td colspan="2">&#160;&#160;&#160;&#160;&#160;&#160; Non-commerical antibody produced in our lab</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axiovert 200M Microscope with Incubating Chamber</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing non-lytic exocytosis of cryptococcus neoformans from macrophages using digital light microscopy

Cryptococcus neoformans ile makrofajların enfeksiyonun pek çok yönü, geniş olarak araştırılmış ve iyi bir şekilde tanımlanmıştır. Ancak, açıkça anlaşılmış değildir belirli bir etkileşim olmayan litik ekzositoz olduğunu. Bu işlemde, maya hücreleri, makrofajlar ve Cn uygulanabilir hem yaprak, az anlaşılmış bir mekanizma ile hücre dışı alana salınır. Burada, zaman lapsed mikroskobu ile 24 saatlik bir enfeksiyon dönemi için ayrı ayrı enfekte makrofajlar çok sayıda takip açıklanmıştır. Enfekte olmuş makrofajların, bir hücre kültürü inkübatöründe olduğu gibi aynı şartlar sağlayan bir mikroskop bağlı bir CO2 atmosfere sahip bir ısıtma odasına yerleştirilmiştir. Canlı dijital mikroskopi statik görüntülerin mevcut olmayan bir ev sahibi ve patojen arasındaki dinamik etkileşimleri hakkında bilgi verebilir. Tersi makrofajlar mantar enfeksiyonları nasıl idare için ipuçları sağlayabilir ve her enfekte hücreyi görselleştirmek için güçlü olmak. Bu teknik, ıkarmaşık bir fenomenin arkasında dinamikleri okuyan sa güçlü bir araçtır.

Bu tekniğin ürettiği görüntüler hücreleri C fagosite sonra ne çevreleyen bilgi toplamak için çeşitli şekillerde analiz edilebilir neoformans. Şekil 1&#39;de görüldüğü gibi, enfekte edilmiş veya enfekte edilmemiş ya da yaklaşık 100 makrofajlar vardır. Bu makrofajlar her biri izleyiciye çok mikroskobik düzeyde konak-patojen etkileşimleri çalışmak için bir fırsat verir. Şekiller 2 ve 3&#39;ün gibi, farklı bir makrofaj ve maya hücre aras?...

Watch this Scientific Journal Video about Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy at JoVE.com

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

Biz makrofaj C görselleştirmek için nasıl açıklar neoformans (Cn) dijital ışık mikroskobu kullanılarak litik olmayan ekzositoz sürecine özel vurgu ile gerçek zamanlı etkileşim,. Ayrı ayrı enfekte makrofajlar bu tekniği kullanarak bu olayın çeşitli yönlerini belirlemek için ele alınabilir.

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 ...

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Immunology and Infection

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

11.7K Görüntüleme.  Albert Einstein College of Medicine.  Biz makrofaj C görselleştirmek için nasıl açıklar neoformans (Cn) dijital ışık mikroskobu kullanılarak litik olmayan ekzositoz sürecine özel vurgu ile gerçek zamanlı etkileşim,. Ayrı ayrı enfekte makrofajlar bu tekniği kullanarak bu olayın çeşitli yönlerini belirlemek için ele alınabilir. 

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

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

HIV ve Live Konfokal Mikroskop Floresan Klonlar kullanılarak HIV görselleştirme Hücre-hücre Transferi

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

İmmünoloji ve Enfeksiyon

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

Eşodaklı Mikroskopi İzole Alveoler Makrofagların anti-fungal Hareketi değerlendirilmesi

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

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

Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFN&#947;) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity.

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

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

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

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

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

Mast Hücre Salgı granüller incelenmesi; biyosentezi adlı ekzositozu için

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

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 ...

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila express gustatory receptors1,2, ion channels3-6, and ionotropic receptors7 that are involved in the detection of volatile and non-volatile sensory cues. These neurons directly contact tastants such as food, noxious substances and pheromones and therefore influence many complex behaviors such as feeding, egg-laying and mating. Electrode recordings and calcium imaging have been widely used in insects to quantify the neuronal responses evoked by these tastants. However, electrophysiology requires specialized equipment and obtaining measurements from a single taste sensillum can be technically challenging depending on the cell-type, size, and position. In addition, single neuron resolution in Drosophila can be difficult to achieve since taste sensilla house more than one type of chemosensory neuron. The live calcium imaging method described here allows responses of single gustatory neurons in live flies to be measured. This method is especially suitable for imaging neuronal responses to lipid pheromones and other ligand types that have low solubility in water-based solvents.

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

Fizyolojik Yanıtları Ölçüm<em&gt; Drosophila</em&gt; Canlı Kalsiyum Görüntüleme kullanma Lipit Feromonlar Sensory Nöronlar

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

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

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

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

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

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

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

Makrofaj hücre dışı tuzaklar Confocal mikroskobu kullanılarak görüntülenmesi

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimuriumyalıtım-makrofajlar üzerinden Phagosomes içeren

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Hücre içi SNARE mikroskobu Imaging Floresans ömür tarafından kaçakçılığı görüntülenmesi

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...