Name: phagocytosis assay for apoptotic cells in drosophila embryos
Uploaded: 2017-08-03T14:00:00
Description: Watch this Scientific Journal Video about Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos at JoVE.com

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...

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engulfment+of+apoptotic+cells+in+C.+elegans+is+mediated+by+integrin+alpha/SRC+signaling.">Engulfment of apoptotic cells in C. elegans is mediated by integrin alpha/SRC signaling.</a> Curr Biol. 20 (6), 477-486 (2010).</li><li>Hanayama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+factor+that+links+apoptotic+cells+to+phagocytes.">Identification of a factor that links apoptotic cells to phagocytes.</a> Nature. 417 (6885), 182-187 (2002).</li><li>Tepass, U., Fessler, L. I., Aziz, A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+of+hemocytes+and+their+relationship+to+cell+death+in+Drosophila.">Embryonic origin of hemocytes and their relationship to cell death in Drosophila.</a> Development. 120 (7), 1829-1837 (1994).</li><li>Gold, K. S., Bruckner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+and+cellular+immunity+in+Drosophila+melanogaster.">Macrophages and cellular immunity in Drosophila melanogaster.</a> Semin Immunol. 27, 357-368 (2015).</li><li>Abrams, J. M., White, K., Fessler, L. I., Steller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+cell+death+during+Drosophila+embryogenesis.">Programmed cell death during Drosophila embryogenesis.</a> Development. 117 (1), 29-43 (1993).</li><li>Franc, N. C., Heitzler, P., Ezekowitz, R. A., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+croquemort+in+phagocytosis+of+apoptotic+cells+in+Drosophila.">Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila.</a> Science. 284 (5422), 1991-1994 (1999).</li><li>Mukae, N., Yokoyama, H., Yokokura, T., Sakoyama, Y., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+innate+immunity+in+Drosophila+by+endogenous+chromosomal+DNA+that+escaped+apoptotic+degradation.">Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation.</a> Genes Dev. 16 (20), 2662-2671 (2002).</li><li>Manaka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Draper-mediated+and+phosphatidylserine-independent+phagocytosis+of+apoptotic+cells+by+Drosophila+hemocytes/macrophages.">Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages.</a> J Biol Chem. 279 (46), 48466-48476 (2004).</li><li>Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: A Practical Approach. , 3868-3878 (1998).</li><li>Kuraishi, T., 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=Pretaporter,+a+Drosophila+protein+serving+as+a+ligand+for+Draper+in+the+phagocytosis+of+apoptotic+cells.">Pretaporter, a Drosophila protein serving as a ligand for Draper in the phagocytosis of apoptotic cells.</a> Embo j. 28 (24), 3868-3878 (2009).</li><li>Franc, N. C., Dimarcq, J. L., Lagueux, M., Hoffmann, J., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Croquemort,+a+novel+Drosophila+hemocyte/macrophage+receptor+that+recognizes+apoptotic+cells.">Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells.</a> Immunity. 4 (5), 431-443 (1996).</li><li>Kuraishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+calreticulin+as+a+marker+for+phagocytosis+of+apoptotic+cells+in+Drosophila.">Identification of calreticulin as a marker for phagocytosis of apoptotic cells in Drosophila.</a> Exp Cell Res. 313 (3), 500-510 (2007).</li><li>Nagaosa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+betanu-mediated+phagocytosis+of+apoptotic+cells+in+Drosophila+embryos.">Integrin betanu-mediated phagocytosis of apoptotic cells in Drosophila embryos.</a> J Biol Chem. 286 (29), 25770-25777 (2011).</li><li>Nonaka, S., Nagaosa, K., Mori, T., Shiratsuchi, A., Nakanishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+alphaPS3/betanu-mediated+phagocytosis+of+apoptotic+cells+and+bacteria+in+Drosophila.">Integrin alphaPS3/betanu-mediated phagocytosis of apoptotic cells and bacteria in Drosophila.</a> J Biol Chem. 288 (15), 10374-10380 (2013).</li><li>Fujita, Y., Nagaosa, K., Shiratsuchi, A., Nakanishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NPxY+motif+in+Draper-mediated+apoptotic+cell+clearance+in+Drosophila.">Role of NPxY motif in Draper-mediated apoptotic cell clearance in Drosophila.</a> Drug Discov Ther. 6 (6), 291-297 (2012).</li><li>Bruckner, K., 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=The+PDGF/VEGF+receptor+controls+blood+cell+survival+in+Drosophila.">The PDGF/VEGF receptor controls blood cell survival in Drosophila.</a> Dev Cell. 7 (1), 73-84 (2004).</li><li>Nonaka, S., 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=Signaling+Pathway+for+Phagocyte+Priming+upon+Encounter+with+Apoptotic+Cells.">Signaling Pathway for Phagocyte Priming upon Encounter with Apoptotic Cells.</a> J Biol Chem. 292 (19), 8059-8072 (2017).</li><li>Hanayama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmune+disease+and+impaired+uptake+of+apoptotic+cells+in+MFG-E8-deficient+mice.">Autoimmune disease and impaired uptake of apoptotic cells in MFG-E8-deficient mice.</a> Science. 304 (5674), 1147-1150 (2004).</li></ol>

<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 ...

The overall goal of this procedure is to measure in vivo phagocytosis in Drosophila to assess the involvement of specific genes of interest in apoptotic cell engulfment. This method can help answer key questions in the immunology and developmental biology fields about the mechanisms involved in apoptotic cell engulfment. The main advantage of this technique is that the cells can be used easily and precisely measured in vivo phagocytosis levels.The implication of this technique extend toward therapy of inflammatory disorders and autoimmune disease as apoptotic cell by phagocytosis is necessary for removing dangerous cells that cause inflammation. Begin by adding 200 adult female and 200 adult male fruit flies and a plate of fresh grape juice agar on yeast into a 50 milliliter conical tube. Close the tube with a sponge cap and incubate the flies in the light at 16 degrees Celsius for two to three days.At the end of the incubation, move the flies to 25 degrees Celsius in the dark for one hour and replace the old plate with a new plate without yeast. Allow the flies to lay eggs for two hours. Then incubate the plate at 16 degrees Celsius for 26 hours.The next day, use a paint brush to transfer the embryos into one milliliter of PBS supplemented with 0.2%Triton X-100. After two washes, add 1.2 milliliters of sodium hypochlorite solution to the embryos to remove the chorion. After three minutes, wash the embryos four times in fresh PBS plus Triton X-100.Transfer the washed embryos onto a six centimeter agarose plate and place the plate under a dissecting microscope. Identify stage 16 embryos using a micro tip. Use a micro pipette to transfer approximately 50 of the stage 16 embryos into a 1.5 milliliter micro test tube.To isolate the embryonic cells, first wash the embryos two times with 150 microliters of PBS. Transfer the embryos into a new 1.5 milliliter micro test tube. Then add 200 microliters of collagenase and homogenize the embryos 30 times with a pellet mixer.At the end of the homogenization, triturate the embryonic cells 10 times and transfer the cell suspension into a new 1.5 milliliter tube. Incubate the cells at 37 degrees Celsius for one minute. Then triturate the cells 10 more times and return them to the 37 degrees Celsius incubator for another minute.At the end of the second incubation, add 800 microliters of PBS to the cells and collect the cells by centrifugation. Resuspend the pellet in 200 microliters of Trypsin before filtering the cell suspension through a 70 micron cell strainer. Stop the enzymatic activity with 40 microliters of heat inactivated fetal bovine serum in 800 microliters of PBS and collect the cells by centrifugation.Resuspend the precipitated cells in 200 microliters of fresh PBS and collect the cells with another centrifugation. Resuspend the pellet in 30 microliters of PBS and mount the cells onto an aminopropyltriethoxysilane-coated glass slide. When the cells have attached, use a pipette to remove the excess PBS from the slide and fix the cells with 60 to 70 microliters of 4%paraformaldehyde.After 10 to 15 minutes, remove the fixative and wash the cells in PBS for one minute. To immunostain the cells with anti-Croquemort antibody, first serially soak the slide in methanol then PBS supplemented with 0.2%Triton X-100 followed by PBS alone. Next, block the nonspecific binding with 20 microliters of 5%whole swine serum in PBS plus 0.2%Triton X-100 for 20 minutes at room temperature.Remove the excess blocking solution and label the cells with 20 microliters of anti-Croquemort antiserum at four degrees Celsius overnight. The next morning, wash the slide in PBS plus Triton X-100 for five 10-minute incubations. After the last wash, rinse the slide in PBS for 10 minutes.Then label the cells with 20 microliters of alkaline phosphatase-labeled Anti-Rat IgG at room temperature for one hour. At the end of the incubation, wash the slide five times in PBS plus Triton X-100 as demonstrated followed by a 10-minute soak in buffer solution. Then label the cells with phosphatase substrate solution and observe the cells under a light microscope.When strong purple signals appear in the hemocyte granules, remove the excess substrate and soak the slide in buffer supplemented with EDTA for five to 10 minutes. At the end of the incubation, rinse the cells with two five-minute PBS washes and treat them with equilibration buffer for 10 minutes. After removing the solution, label the cells with 20 microliters of terminal deoxynucleotidyl transferase solution at 37 degrees Celsius for one hour.Then remove the solution from the slide and soak the cells in 0.5 milliliters stop wash buffer in 17 milliliters of water for 10 minutes. Next, rinse the cells with three five-minute PBS washes and label them with 20 microliters of anti-digoxigenin peroxidase for 30 minutes at room temperature. At the end of the incubation, wash the cells four times in PBS as demonstrated and soak the slide in peroxidase substrate for 30-second increments until the apoptotic cells turn brown.When an optimal staining has been achieved, soak the slide in water to stop the peroxidase reaction. In these images, Drosophila macrophages called hemocytes were stained for the hemocyte marker Croquemort and for the presence of phagocytosed apoptotic cells by TUNEL in dispersed embryonic cells as just demonstrated. Croquemort positive cells exhibit purple signals in the small granules of their cells while TUNEL positive cells demonstrated brown signal in their whole corpuses.Alternatively, hemocytes can be stained with an anti-GFP antibody which stains the entire hemocyte instead of just the granules. In this experiment, the ratio of phagocytosing hemocytes to total hemocytes in wild type or single mutant embryos was analyzed, demonstrating that the phagocytic index was lower in both single mutant strains than in wild type flies while the total numbers of hemocytes and apoptotic cells were similar indicating the requirement of the mutated genes for apoptotic cell engulfment. RNAi knockdown of single genes also reduces the phagocytic index while the total numbers of hemocytes and apoptotic cells remained comparable, further underscoring the involvement of the investigated phagocytosis receptors in apoptotic cell mediated phagocytosis.Once mastered, this technique can be completed in about 15 hours if it is performed properly. While attempting this procedure, it's important to remember to avoid excess hemocyte marker and TUNEL staining for optimal evaluation of the phagocytotic ability of the cells. Following this procedure, in situ phagocytosis assay of all Drosophila embryos can be performed to answer additional questions about the site of engulfment or the distribution of the hemocytes.After its development, this technique paved the way for researchers in the immunology field to explore the mechanisms of apoptotic cell engulfment in Drosophila. After watching this video, you should have a good understanding of how to measure in vivo phagocytosis in Drosophila embryos.

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Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

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