Onların tavsiyeleri için Kaz Nagaosa ve Akiko Shiratsuchi&#39;ye minnettarız.

1. Hazırlık <ol><li> Taze üzüm suyu agar plakalarının hazırlanması <ol><li> 4.4 g agarda 100 mL su ilave edin ve karışımı mikrodalga fırında ısıtarak agarı çözün. </li><li> 80 mL taze üzüm suyu, 5 mL asetik asit ve 5 mL etanol, agar solüsyonuna ilave edin. </li><li> Her cam slayt üzerine bir pipet ile yaklaşık 1.5 mL çözelti dökün ve katılaşmasına izin verin. </li></ol></li><li> 6 cm çanak agaroz plakalarının hazırlanması <ol><li> 0.5 g agaroza 50 mL su ilave edin ve agarozu çözmek için mikrodalga fırında ısıtın. </li><li> Bir pipet ile 6 cm&#39;lik bir tabağa yaklaşık 2.5 mL agaroz çözeltisi dökün. </li></ol></li></ol> 2. Evre 16 Embriyo Koleksiyonu <ol><li> Yetişkin sinekler toplayın ve 200 mL dişiler ve 200 erkek bir 50 mL konik tüp ekleyin. </li><li> Maya üzerine taze üzüm suyu agarı bir tabak 50 mL tüp içine koyun ve bir sünger cas. </li><li> Sinekleri ışık altında 16 ° C&#39;de 2 - 3 gün inkübe edin. Plakayı günde bir kez değiştirin. </li><li> Sinekleri karanlıkta 25 ° C&#39;de 1 saat hareket ettirin. </li><li> Eski tabağı mayasız yeni bir tabakaya değiştirin. Uçaklara 2 saat yumurta bırakmasına izin ver. </li><li> Plakayı toplayın ve 16 saat boyunca inkübe edin. 26 saat süreyle inkübe edin. </li><li> % 0.2 (v / v) Triton X-100 içeren 1 mL PBS içine bir boya fırçası ile plakadan embriyolar toplayın. </li><li> Embriyoları,% 0.2 (v / v) Triton X-100 içeren 1 mL PBS ile iki kez yıkayın. </li><li> Porsiyonu çıkartmak için, 3 dakika embriyoya 1.2 mL sodyum hipoklorit çözeltisi (% 2.2 - 3.4 Cl) ekleyin. </li><li> Embriyoları,% 0.2 (v / v) Triton X-100 içeren 1 mL PBS ile 4 kez yıkayın. </li><li> 6 cm çanak agaroz plakaları embriyolar yerleştirin. Evre 16 embriyoları, Roberts 20 tarafından tarif edildiği gibi mikroskop altında alın. Bir mikropipet ile 1.5 mL&#39;lik bir Treff mikro test tüpünde yaklaşık 50 embriyo toplayın. </li></ol> 3. PrepaEmbriyonik Hücrelerin Rasyonları <ol><li> Embriyoları iki kez 150 μL PBS ile yıkayın. </li><li> PBS içinde kollajenaz 200 uL (% 0.25 (w / v)) varlığında embriyoları 1.5 mL mikro test tüpü ve pelet mikserinde 30 kez homojenize edin. </li><li> Embriyonik hücreleri 10 kez pipetle dağıtın ve hücre süspansiyonunu 1.5 mL tüp içine alın. Hücreleri 37 ° C&#39;de 1 dakika su banyosunda inkübe edin. Bu adımı iki kez tekrarlayın. </li><li> Hücre süspansiyonuna 800 μL PBS ekleyin ve 4 ° C&#39;de 1400 x g&#39;de 5 dakika santrifüjleyin. </li><li> Süpernatantı çıkarın ve hücrelere PBS içinde 200 μL tripsin (% 0.25 (w / v)) ekleyin. Embriyonik hücreleri 50 kez pipetle dağıtın. </li><li> Hücre süspansiyonunu 70 um&#39;lik bir Hücre Süzücü ile süzün. </li><li> Tripsin aktivitesini durdurmak için filtrelenmiş hücre süspansiyonuna 40 μL ısı ile inaktive fetal sığır serumu ekleyin. 800 μL PBS ekleyin ve 4 ° C&#39;de 1,400 x g&#39;de 5 dakika süreyle santrifüjleyin. </li><li> Supernata&#39;yı kaldırNt, 200 ul PBS içinde çökelen hücreleri askıya alın ve 1400 x g&#39;de 4 ° C&#39;de 5 dakika santrifüjleyin. </li><li> Süpernatantı çıkarın ve 30 uL PBS içinde çökelen hücreleri bekletin. </li><li> Hazırlanan hücre süspansiyonunu aminopropil trietoksisilan kaplı cam slaytlara monte edin ve hücreler cam slaytlara tutturuncaya kadar 10-15 dakika bekleyin. </li><li> Cam slaytlardaki kalan çözeltiyi bir pipetle çıkarın. Tespit için 10-15 dakika boyunca hücrelere% 4 (w / v) PFA (paraformaldehid) içeren PBS&#39;nin 60 - 70 μL&#39;sini monte edin. </li><li> Sabitleme çözeltisini çıkarın ve 1 dakikadan fazla cam kaydırıcıları PBS&#39;ye yerleştirin. </li></ol> 4. Hemocytes Boyama <ol><li> Bir anti-Croquemort antikoru ile immün boyama <ol><li> 10 dakika boyunca% 0,2 (v / v) Triton X-100 içeren PBS içinde ve sonra 10 dakika boyunca PBS&#39;de, 10 dakika boyunca metanol içinde cam kaydırıcıları ıslatınız. </li><li> PBS içerisinde 20 μL% 5 (v / v) tam domuz serumuEngelleme hücreleri üzerinde% 0.2 (v / v) Triton X-100. Slaytları 20 dakika boyunca oda sıcaklığında inkübe edin. </li><li> Çözeltiyi cam kaydıraklardan çıkarın ve hücrelere% 0.2 (v / v) Triton X-100 içeren PBS içinde 20 μL anti-Croquemort antiserumu 19 , 21 (% 0.1 (v / v)) yerleştirin. Slaytları gece boyunca 4 ° C&#39;de inkübe edin. </li><li> 10 dakika boyunca 5 kez% 0.2 (v / v) Triton X-100 içeren PBS içinde cam sürgüleri bekletin. </li><li> Cam kaydırakları 10 dakika PBS&#39;de bekletin. </li><li> Odalarda hücrelere% 0.2 (v / v) Triton X-100 ve% 5 (v / v) bütün domuz serumunu içeren PBS içinde 20 uL alkalin fosfataz etiketli anti-sıçan IgG&#39;si (% 0.5 v / v) Sıcaklık 1 saat. </li><li> 10 dakika boyunca 5 kez% 0.2 (v / v) Triton X-100 içeren PBS içinde cam sürgüleri bekletin. </li><li> 100 mM Tris-HCl, pH 9.5, 100 mM NaCl ihtiva eden tampon içinde cam slaytlar ıslatın ve 10 dakika için 50 mM MgCl2. </li><li> 0.23 mg / mL 5-bromo-4 içeren dağ fosfataz substrat çözeltisi-kloro-3-indolil-fosfat (BCIP), 0.35 mg / mL nitrotetrazolivm (NBT), 100 mM Tris-HCI, pH 9.5, 100 mM NaCI, ve hücreler 50 mM MgCl2. </li><li> Düzgün boyanması için hücreleri mikroskop altında izleyin. Hemocytes granüllerinde güçlü mor sinyalleri göründüğünde, substrat solüsyonu çıkarın ve 10 mM Tris-HC1, pH 8.5 ve 1 mM EDTA içeren tampon cam slaytlar batırın. Yaklaşık 5 - 10 dakika boyanması önerilir. </li></ol></li><li> Bir anti-GFP antikoru ile immün boyama (hemositlerin boyanması için başka bir seçenek) <ol><li> Cam kaydırakları 10 dakika süreyle metanol içinde, 10 dakika için% 0.2 (v / v) Triton X-100 ve 10 dakika PBS içeren PBS&#39;yi ıslatınız. </li><li> Blokaj için hücrelere% 0.2 (v / v) Triton X-100 içeren PBS içinde% 5 (v / v) tam domuz serumu 20 uL&#39;lik bir miktarı monte edin. Slaytları 20 dakika boyunca oda sıcaklığında inkübe edin. </li><li> Cam slaytlardaki çözeltiyi çıkarın ve 20 μL fare anti-GFP antikoru (% 1 (v / v)) / PBS cHücrelere% 0.2 (v / v) Triton X-100 ile ulaşılmaktadır. Slaytları gece boyunca oda sıcaklığında inkübe edin. </li><li> 10 dakika boyunca 5 kez% 0.2 (v / v) Triton X-100 içeren PBS içinde cam sürgüleri bekletin. </li><li> Cam kaydırakları 10 dakika PBS&#39;de bekletin. </li><li> Odalarda hücrelere% 0.2 (v / v) Triton X-100 ve% 5 (v / v) tüm domuz serumu içeren PBS&#39;de 20 uL alkalin fosfataz etiketli anti-fare IgG&#39;si (% 0.5 v / v) Sıcaklık 1 saat. </li><li> 10 dakika boyunca 5 kez% 0.2 (v / v) Triton X-100 içeren PBS içinde cam sürgüleri bekletin. </li><li> 100 mM Tris-HCI, pH 9.5, 100 mM NaCl&#39;den oluşan tampon içinde cam slaytlar ıslatın ve 10 dakika için 50 mM MgCl2. </li><li> 0.23 mg / ml BCIP, 0.35 mg / ml NBT, 100 mM Tris-HCl, pH 9.5, 100 mM NaCI, ve hücreler 50 mM MgCl2 ihtiva eden fosfataz alt-tabaka çözeltisi monte edin. </li><li> Düzgün boyanması için hücreleri mikroskop altında izleyin. Tam hemositlerde mor sinyaller göründüğünde, substrat solüsyonunu çıkarın ve içindeki slaytları,10 mM Tris-HC1, pH 8.5 ve 1 mM EDTA. Yaklaşık 10-15 dakika boyanması önerilir. </li></ol></li></ol> 5. TUNEL tarafından Apoptotik Hücrelerin Boyanması <ol><li> Cam kaydırakları 5 dakika PBS&#39;de bekletin. </li><li> Yeni PBS ile tekrarlayın. </li><li> Dengeleyici dengeleyici tamponu (Malzeme Tablosuna bakınız) 10 dakika boyunca hücrelerde. </li><li> Cam slaytlar solüsyonu kaldırın ve 1 saat 37 ° C&#39;de hücrelere 15 uL Reaksiyon Tamponu ve terminal deoksinükleotidil transferaz 5 mcL içeren TdT çözeltisi 20 mcL monte edin. </li><li> Çözeltiyi cam kaydıraklardan çıkarın ve slaytları 0.5 mL STOP / Yıkama tamponu ve 17 mL sudan oluşan tampona batırın. </li><li> Slaytları PBS içinde 5 dakika 3 kez bekletin. </li><li> 30 dk boyunca oda sıcaklığında hücrelere 20 μL Anti-Digoxigenin-Peroksidaz ekleyin. </li><li> Cam kaydırakları 5 dakika 4 kez PBS&#39;de bekletin. </li><li> Cam slaytları 30 mL 50 mM Tris-HCl, pH 7.5 suşu içeren peroksidaz substrat çözeltisine batırın30 sn için 3,3&#39;-diaminobenzidin tetrahydrichloride (DAB) ve% 0.002 (h / h) H2 O 2 ga tam mikro spatula. </li><li> Apoptotik hücreler kahverengi oluncaya dek adım 5.9&#39;u tekrarlayın. Peroksidaz reaksiyonunu durdurmak için cam kaydırıcıları suya batırın. </li><li> Gözlem için hücreleri su ile kapatın. </li></ol> 6. Apoptotik Hücrelerin Fagositoz Düzeyini ölçün <ol><li> Fagositoz düzeyini değerlendirmek için örnekleri hafif bir mikroskopta izleyin. Hemocytes olarak Croquemort pozitif hücreleri veya GFP pozitif hücreleri, Apoptotik hücreler olarak TUNEL pozitif hücreleri ve hemositleri fagositize eden çift pozitif hücreleri sayın. </li><li> Fagositik indeks, Croquemort pozitif hücrelerin veya GFP pozitif hücrelerin toplam sayısına karşı çift pozitif hücre sayısı olarak tanımlanır. 300&#39;den fazla hemosit izlenmesi önerilir. </li></ol>

<ol>
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Yazarlar, rekabet eden mali çıkarlarının olmadığını beyan ettiler.

<html> Burada, Drosophila embriyolarını kullanan bir fagositoz tahlili tarif ettik. Fagositozu niceliksel olarak ölçmek için dağınık embriyonik hücreler kullanarak Hemocytes, Drosophila professional phagocytes, Hemocyte marker Croquemort veya srpHemo- kaynaklı GFP için immünostained ve apoptotik hücreler bu protokolde TUNEL tarafından tespit edilir. Fagositoz seviyesi, hemositlerin ve fagosit oksitleyici hemositlerin toplam sayısını sayarak bir fagositik indeks olarak ifade edi...

Metazoan hayvanlarda, örneğin nematod Caenorhabditis elegans , meyve sinek Drosophila melanogaster ve fare ve insanlar, çok sayıda hücre vücutlarını şekillendirmek için gelişme döneminde ve erişkinlikte homeostazı 1 , 2 korumak için apoptoz geçirir. Apoptotik hücrelerin hızla çıkartılması gerekir çünkü bunlar, tamamen çıkarılmasa bile, immünojenik hücre içi materyalleri serbest bırakarak çevreleyen dokularda iltihap oluştururlar 3 . Hızlı kaldırmayı kolaylaştırmak için, apoptotik hücreler, fagositlerin yutulma reseptörleri tarafından tanınan ve yememe sinyalleri olarak adlandırılırlar ve fagositoz 3 , 4 , 5 , 6 ile ortadan kaldırılırlar. Böylece, fagositoz ana homeostazın korunmasında hayati bir rol oynar ve bu nedenle molekülün aydınlatılması<html> Apoptotik hücrelerin fagositozunun altında yatan mekanizmalar önemlidir. Apoptotik hücrelerin fagositozundan sorumlu mekanizmalar, nematodlar, sinekler ve farelerdeki türler arasında evrimsel olarak korunmuş gibi gözükmektedir 7 . Bu model hayvanların apoptotik hücrelerin soğumasını değerlendirmek için çeşitli fagositoz testleri mevcuttur. C. elegans içinde, 131 somatik hücre gelişimi sırasında programlanmış hücre ölümü ve hücre cesetleri profesyonel olmayan fagositler 8 olan komşu hücreler tarafından fagosite edilen. Böylece, C. elegans&#39;taki kalan hücre cesetlerinin sayısının sayılması, in vivo fagositoz seviyesini gösterir. Ölü hücrelerin artan sayısını gösteren nematod mutantlarını araştırarak, fagositoz için gereken birkaç gen tespit edildi ve genetik olarak 9 , 10 ,S = &quot;xref&quot;&gt; 11 , 12 . Birincil kültür fagositleri (genellikle makrofajlar) ile ex vivo fagositoz testleri, farelerde sıklıkla kullanılır. Apoptotik hücreler, Jurkat hücreleri gibi hücre hatları kullanılarak hazırlanır ve primer fagositlerle karıştırılır. Birkaç saat inkübasyondan sonra fagositoz seviyesini değerlendirmek için toplam fagosit sayısı ve sindirim fagosit sayısı hesaplanır. Bu yöntemin sofistike bir modifikasyonu olan Nagata&#39;nın grubu, apoptotik hücrelerin apoptotik DNA fragmantasyonuna maruz kalmadığı, ancak kaspaza dirençli bir ICAD (kaspazla aktive DNaz inhibitörü) eksprese eden hücrelerle ex vivo fagositoz testi geliştirdi; ancak DNA hala kesildiğinde Hücreler fagositozlanır. Bu hücreler, bir fagositoz tahlilinde apoptotik hedefler olarak kullanıldığında, sadece sarmalanmış apoptotik hücrelerin DNA&#39;sı parçalanır ve TdT aracılı dUTP nick uç etiketlemesi (TUNEL) ile boyanır. Bu nedenle, leveL apoptotik hücre yutulması, bir fagosit ve apoptotik hücre karışımı 13 içinde TUNEL sinyallerinin sayılmasıyla ölçülür. D. melanogaster&#39;da , hemocytes adında profesyonel fagositler, Drosophila makrofajları, 14 , 15 apoptotik hücrelerin fagositozundan sorumludur. Kültür hücresi soyları ile yapılan in vitro fagositoz analizlerine ek olarak , bütün Drosophila embriyolarıyla in vivo fagositoz analizleri mevcuttur. Drosophila embriyolar birçok hücreleri apoptozise uğrar ve embriyonik gelişim 14, 15, 16 boyunca hemocytes tarafından fagosite edilir çünkü apoptotik hücre yutulma seviyesinin incelenmesine yönelik güçlü bir araçtır. Bir in vivo fagositoz tahliline bir örnek, Franc&#39;in grubu tarafından geliştirilen yöntemdir. Yöntemlerinde hemositler deBir hemosit işareti olan peroksidazenin immüno-lekelemesi ile tespit edilen apoptotik hücreler, bütün Drosophila embriyolarında 7-amino aktinomisin D nükleer boya kullanılarak lekelenmiş ve çift pozitif hücrelerin sayısı, fagositoz 17 sinyali olarak sayılmıştır. Embriyolarda bir fagositoz tahlili için bir başka örnek, yukarıda tarif edilen Nagata metodu kavramına dayanır; Bununla birlikte, in vivo fagositoz, dCAD ( Drosophila caspase-activated DNase) mutant sinekler 18 , 19&#39;un embriyoları kullanılarak değerlendirilir. Bu in vivo fagositoz testleri doğrudan in situ fagositoz gözlemlemek için yararlıdır. Bununla birlikte, zorluklar, fagosit hücrelerini sayma aşamasındaki muhtemel yanlılıkların hariç tutulması ile ilişkilidir; çünkü kalınlığından ötürü bütün embriyoların tüm fagositlerini ve apoptotik hücrelerini gözlemlemek zordur. Bu kısıtlamanın üstesinden gelmek için,Drosophila embriyolarında yeni bir fagositoz testi yaptı. Yöntemimizde, fagositozan hemositleri kolayca sayabilmek için, bütün embriyolar dağınık embriyonik hücreleri hazırlamak için homojenize edilir. Fagositler, bir fagosit markörünün immün boyaması ile tespit edilir ve apoptotik hücreler, bu dağınık embriyonik hücreler ile TUNEL tarafından saptanır. Dağıtılan embriyonik hücrelerin kullanılması, in vivo fagositoz düzeylerini, fagositoz düzeyini tam olarak ölçen bir in vitro fagositoz tahlilini gerçekleştirmiş gibi ölçmenizi sağlar. Bunlar embriyo 20 16 fagositozla olan apoptotik hücre mesafesi en bol gelişimsel aşama aşama için gelişirse sinekler tüm genotipler Bu deneyde kullanılabilir. Bu yöntem, fagositoz seviyesini kantitatif olarak değerlendirmenin avantajına sahiptir ve bu nedenle apoptotik hücrelerin in vivo fagositozunda yer alan yeni moleküllerin tanımlanmasına katkıda bulunabilir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>whole swine serum</td>
			<td>MP Biomedicals</td>
			<td>55993</td>
			<td>For bloking</td>
		</tr>
		<tr>
			<td>Treff micro test tube(easy fit)&#160; Dnase, Rnase free tube, 1.5 mL</td>
			<td>TreffLab</td>
			<td>96. 4625. 9. 01</td>
			<td>For&#160; homogenization</td>
		</tr>
		<tr>
			<td>pellet mixer&#160; 1.5 mL</td>
			<td>TreffLab</td>
			<td>96. 7339. 9. 03</td>
			<td>For&#160; homogenization</td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma-Aldrich</td>
			<td>C-0130</td>
			<td>For preparation of embryonic cells</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Fisher SCIENTIFIC</td>
			<td>27250-018</td>
			<td>For preparation of embryonic cells</td>
		</tr>
		<tr>
			<td>Kpl Anti-Rat IgG (H+L) Ab MSA, AP</td>
			<td>KPL</td>
			<td>475-1612</td>
			<td>secondary antibody for 
			stainig hemocytes with an anti-Croquemort antibody</td>
		</tr>
		<tr>
			<td>5-bromo-4-chloro- 
			3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11383221001</td>
			<td>BCIP, For staining of hemocytes</td>
		</tr>
		<tr>
			<td>nitro blue tetrazolium</td>
			<td>Roche</td>
			<td>11383213001</td>
			<td>NBT, For staining of hemocytes</td>
		</tr>
		<tr>
			<td>Anti-Croquemort antibody</td>
			<td></td>
			<td></td>
			<td>described previously in Manaka et al, J. Biol. Chem., 279, 48466-48476</td>
		</tr>
		<tr>
			<td>&#160;Anti-GFP 
			from mouse IgG1&#954; (clones 7.1 and 13.1)&#160;</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td>For staining of hemocytes</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG-AP Conjugate</td>
			<td>Bio-Rad</td>
			<td>170-6520</td>
			<td>secondary antibody for stainig hemocytes with an anti-Croquemort antibody</td>
		</tr>
		<tr>
			<td>Apop Tag Peroxidase In Situ Apoptosis Detection Kit</td>
			<td>Millipore</td>
			<td>S7100</td>
			<td>For staining of apoptoitc cells. This kit includes Equilibration buffer, Reaction buffer, STOP/Wash buffer, TdT enzyme, and Anti-Digoxigenin-Peroxidase.</td>
		</tr>
		<tr>
			<td>3,3&#39;-diaminobenzidine 
			tetrahydrichloride</td>
			<td>nacalai tesque</td>
			<td>11009-41</td>
			<td>DAB, For staining of apoptoitc cells</td>
		</tr>
		<tr>
			<td colspan="2">Table of Fly Strains</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>w1118</td>
			<td></td>
			<td></td>
			<td>Control flies, described in Freeman et al., Neuron, 38, 567-580</td>
		</tr>
		<tr>
			<td>drpr&#916;5</td>
			<td></td>
			<td></td>
			<td>drpr mutant, described in Freeman et al, Neuron, 38, 567-580</td>
		</tr>
		<tr>
			<td>Itgbn2</td>
			<td></td>
			<td></td>
			<td>Itgbn mutant, described in Devenport et al., Development, 131, 5405-5415</td>
		</tr>
		<tr>
			<td>srpHemoGAL4 UAS-EGFP</td>
			<td></td>
			<td></td>
			<td>described in Br&#252;ckner et al., Dev. Cell., 7, 73-84</td>
		</tr>
		<tr>
			<td>UAS-drpr-IR</td>
			<td>VDRC</td>
			<td>4833</td>
			<td>-</td>
		</tr>
		<tr>
			<td>UAS-Itgbn-IR</td>
			<td>NIG-fly</td>
			<td>1762R-1</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

phagocytosis assay for apoptotic cells in drosophila embryos

Apoptotik hücrelerin fagositozunun altında yatan moleküler mekanizmalar, bağışıklık ve inflamatuar dirençli hastalıklarda rolünden dolayı daha ayrıntılı olarak açıklığa kavuşturulmalıdır. Burada, fide soğurumunu kontrol eden gen şebekesinin memelilerden evrimsel olarak korunduğu Drosophila meyve sineği kullanılarak kantitatif olarak fagositozu araştırmak için deneysel bir yöntem geliştirdik. Tam hayvanlar kullanılarak engulfing ve un-engulfing fagositleri doğru tespit etmek ve saymak için Drosophila embriyoları, fagositler ve apoptotik hücreler de dahil olmak üzere dağılmış hücreler elde etmek üzere homojenize edildi. Dağıtılmış embriyonik hücrelerin kullanımı, in vitro fagositoz düzeylerini, bütün embriyolardaki tüm fagositleri ve apoptotik hücreleri gözlemlemek ve fagositoz düzeyini tam olarak ölçmek mümkün olan bir in vitro fagositoz tahlilini gerçekleştirmiş gibi ölçebilir. Bu yöntemin daha önceki çalışmaların yöntemlerini ürettiğini doğruladıkBu, apoptotik hücrelerin fagositozu için gerekli genleri belirledi. Bu yöntem, ölü hücrelerin parçalanmasını sağlar ve Drosophila&#39;nın güçlü genetiği ile birleştirildiğinde, fagositler tarafından apoptotik hücrelerin migrasyonu, tanınması, yutulması ve parçalanması ile oluşan karmaşık fagositik reaksiyonları ortaya çıkaracaktır.

Apoptotik hücrelerin fagositozunu incelemek amacıyla gelişim aşaması 16&#39;nın Drosophila embriyoları toplandı ve dağılmış hücreler olarak hazırlandı. Hemocytes, Drosophila makrofajları, hemocyte marker &quot;Croquemort&quot; 17 , 22 için immünositokimya ile spesifik bir antikor 19 , 21 kullanarak lekelenmiş ve apoptotik hücreler da?...

Watch this Scientific Journal Video about Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos at JoVE.com

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

Drosophila&#39;nın dağıtılmış embriyonik hücrelerini kullanarak bir fagositoz tahlili burada tarif edilmektedir. Bu, in vivo fagositoz seviyelerini kolay ve kesin bir şekilde ölçmemize ve apoptotik hücrelerin fagositozu için gerekli olan yeni molekülleri belirlememize olanak tanır.

Apoptotik Hücreler İçin Fagositoz Testi<em&gt; Drosophila</emEmbriyolar

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

phagocytosis-assay-for-apoptotic-cells-in-drosophila-embryos

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Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

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Cell Death

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9.8K Görüntüleme.  Kanazawa University.  Drosophila'nın dağıtılmış embriyonik hücrelerini kullanarak bir fagositoz tahlili burada tarif edilmektedir. Bu, in vivo fagositoz seviyelerini kolay ve kesin bir şekilde ölçmemize ve apoptotik hücrelerin fagositozu için gerekli olan yeni molekülleri belirlememize olanak tanır. 

Video: Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

RNAi Tarama için ilgilendim Ana Faktörler Vaccinia Virüs Enfeksiyonu Drosophila Hücre

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

İmmünoloji ve Enfeksiyon

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

2-foton Mikroskop kullanarak Enfekte Akciğerler Nötrofiller Fagositoz ve NET-oluşumu Doğrudan Gözlem

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Hücre-kaderi ardından<em&gt; E. coli</em&gt; Faj Lambda tarafından Enfeksiyon Sonrası

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

Nicel<em&gt; In vitro</emStatik Koşullarında Account İlköğretim İnsan mikrovasküler endotel hücreleri için Nötrofil yapışma Tedbir&gt; Testi

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Retina Pigment Epitel Hücreleri üzerinde Fagositoz Tahliller için Domuz veya Sığır fotoreseptör dış Kesimleri Büyük Ölçekli Arıtma

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Bir Monosit Tek tabakalı Testi Kullanımı Fcy reseptör aracılı fagositoz değerlendirin

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Geliştirme ve doğrulama vericiyi bir tek molekül dijital enzim bağlı Immunosorbent Assay insan Interferon-α için dizi

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Alveoler makrofaj fagositoz ve bakteri izni farelerde

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

Bir In Vivo fagositoz tahlil kullanarak Drosophila melanogaster, meyve sineği hücresel bağışıklık yanıtı değerlendirirken

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...