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

Agradecemos a Kaz Nagaosa e Akiko Shiratsuchi pelo seu conselho.

1. Preparação <ol><li> Preparação de placas de agar de suco de uva fresca <ol><li> Adicione 100 mL de água a 4,4 g de ágar e aquecer a mistura num forno de microondas para dissolver o ágar. </li><li> Adicione 80 mL de suco de uva fresco, 5 mL de ácido acético e 5 mL de solução de etanol em ágar. </li><li> Despeje aproximadamente 1,5 ml da solução com uma pipeta em cada lâmina de vidro e deixe solidificar. </li></ol></li><li> Preparação de placas de agarose de prato de 6 cm</st...

<ol>
	<li>Danial, N. N., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death:+critical+control+points.">Cell death: critical control points.</a> Cell. 116 (2), 205-219 (2004).</li><li>Vaux, D. L., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+in+development.">Cell death in development.</a> Cell. 96 (2), 245-254 (1999).</li><li>Savill, J., Fadok, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corpse+clearance+defines+the+meaning+of+cell+death.">Corpse clearance defines the meaning of cell death.</a> Nature. 407 (6805), 784-788 (2000).</li><li>Lauber, K., Blumenthal, S. G., Waibel, M., Wesselborg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+cells:+getting+rid+of+the+corpses.">Clearance of apoptotic cells: getting rid of the corpses.</a> Mol Cell. 14 (3), 277-287 (2004).</li><li>Ravichandran, K. S., Lorenz, U. <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:+signals+for+a+good+meal.">Engulfment of apoptotic cells: signals for a good meal.</a> Nat Rev Immunol. 7 (12), 964-974 (2007).</li><li>Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beginnings+of+a+good+apoptotic+meal:+the+find-me+and+eat-me+signaling+pathways.">Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways.</a> Immunity. 35 (4), 445-455 (2011).</li><li>Nakanishi, Y., Nagaosa, K., Shiratsuchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+removal+of+cells+that+have+become+unwanted:+implications+for+animal+development+and+tissue+homeostasis.">Phagocytic removal of cells that have become unwanted: implications for animal development and tissue homeostasis.</a> Dev Growth Differ. 53 (2), 149-160 (2011).</li><li>Reddien, P. W., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+engulfment+process+of+programmed+cell+death+in+caenorhabditis+elegans.">The engulfment process of programmed cell death in caenorhabditis elegans.</a> Annu Rev Cell Dev Biol. 20, 193-221 (2004).</li><li>Hedgecock, E. M., Sulston, J. E., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+programmed+cell+deaths+in+the+nematode+Caenorhabditis+elegans.">Mutations affecting programmed cell deaths in the nematode Caenorhabditis elegans.</a> Science. 220 (4603), 1277-1279 (1983).</li><li>Ellis, R. E., Jacobson, D. M., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+required+for+the+engulfment+of+cell+corpses+during+programmed+cell+death+in+Caenorhabditis+elegans.">Genes required for the engulfment of cell corpses during programmed cell death in Caenorhabditis elegans.</a> Genetics. 129 (1), 79-94 (1991).</li><li>Gumienny, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CED-12/ELMO,+a+novel+member+of+the+CrkII/Dock180/Rac+pathway,+is+required+for+phagocytosis+and+cell+migration.">CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration.</a> Cell. 107 (1), 27-41 (2001).</li><li>Hsu, T. Y., Wu, Y. 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> Aqui descrevemos um ensaio de fagocitose usando embriões de Drosophila . Ao usar células embrionárias dispersas para medir a fagocitose quantitativamente, os hemócitos, os fagócitos profissionais de Drosophila , são imunossincionados para o marcador de hemicíticos Croquemort ou GFP dirigido por srpHemo , e as células apoptóticas são detectadas por TUNEL neste protocolo. O nível de fagocitose é expresso como um índice fagocítico contando o número total de hemócitos e hemoc...

Em animais metazoários, por exemplo , o nematóide Caenorhabditis elegans , a mosca da fruta Drosophila melanogaster e os camundongos e os seres humanos, um grande número de células sofre apoptose durante o desenvolvimento para moldar seus corpos e na idade adulta para manter a homeostase 1 , 2 . As células apoptóticas precisam ser rapidamente removidas porque induzem inflamação nos tecidos circundantes, liberando materiais intracelulares imunogênicos, se não forem completamente removidos 3 . A fim de facilitar a remoção rápida, as células apoptóticas apresentam os chamados sinais de comer-me que são reconhecidos pelos receptores de engulfment de fagócitos e são eliminados por fagocitose 3 , 4 , 5 , 6 . Assim, a fagocitose desempenha um papel crucial na manutenção da homeostase do hospedeiro e, portanto, elucidando a molécula<html> Os mecanismos subjacentes à fagocitose das células apoptóticas são importantes. Os mecanismos responsáveis ​​pela fagocitose das células apoptóticas parecem ser conservados evolutivamente entre as espécies nos nematóides, moscas e camundongos 7 . Vários ensaios de fagocitose estão atualmente disponíveis para avaliar a absorção de células apoptóticas nestes animais modelo. Em C. elegans , 131 células somáticas sofrem morte celular programada durante o desenvolvimento, e cadáveres celulares são fagocitadas por células vizinhas, que são fagócitos não profissionais 8 . Assim, contar o número de cadáveres de células restantes em C. elegans indica o nível de fagocitose in vivo . Ao procurar por mutantes de nematóides que mostram um número aumentado de células mortas, vários genes necessários para a fagocitose foram identificados e caracterizados geneticamente 9 , 10 ,S = &quot;xref&quot;&gt; 11 , 12 . Os ensaios de fagocitose ex vivo com fagócitos de cultura primária, geralmente macrófagos, são frequentemente utilizados em camundongos. As células apoptóticas são preparadas usando linhas celulares tais como células Jurkat e são misturadas com fagócitos primários. Após uma incubação por várias horas, o número total de fagócitos e fagócitos envolventes são contados para avaliar o nível de fagocitose. Como uma modificação sofisticada deste método, o grupo de Nagata desenvolveu um teste de fagocitose ex vivo com células que expressam um ICAD resistente a caspases (inibidor da DNase ativada por caspase), em que as células apoptóticas não sofrem fragmentação do DNA apoptótico, mas o DNA ainda é escorado quando As células são fagocitadas. Quando estas células são utilizadas como alvos apoptóticos num ensaio de fagocitose, apenas o ADN de células apoptóticas engolidas é fragmentado e corado por marcação de endereçamento DUTP mediada por TdT (TUNEL). Portanto, o leveL de engolimento de células apoptóticas é medido contando sinais de TUNEL em uma mistura de fagócitos e células apoptóticas 13 . Em D. melanogaster , fagócitos profissionais denominados hemócitos, macrófagos de Drosophila , são responsáveis ​​pela fagocitose das células apoptóticas 14 , 15 . Além dos ensaios de fagocitose in vitro com linhas celulares de cultura, estão disponíveis ensaios de fagocitose in vivo com embriões de Drosophila inteiros. Os embriões de Drosophila são uma poderosa ferramenta para examinar o nível de absorção de células apoptóticas porque muitas células sofrem apoptose e são fagocitadas por hemócitos durante o desenvolvimento embrionário 14 , 15 , 16 . Um exemplo de um teste de fagocitose in vivo é o método desenvolvido pelo grupo de Franc. No seu método, os hemócitos são deProtegido pela imunocoloração da peroxidasina, um marcador hemicítico, as células apoptóticas são coradas usando o corante nuclear, a 7-amino-actinomicina D em embriões de Drosophila inteiros, e o número de células positivas duplas é contado como um sinal de fagocitose 17 . Outro exemplo de um teste de fagocitose em embriões baseia-se no conceito do método de Nagata descrito acima; No entanto, a fagocitose in vivo é avaliada utilizando os embriões de DNC ( Drosophila caspase-activada DNase) moscas mutantes 18 , 19 . Estes ensaios de fagocitose in vivo são úteis para observar diretamente a fagocitose in situ . Contudo, as dificuldades estão associadas à exclusão de qualquer possível viés na etapa de contagem de células fagocitantes porque é difícil observar todos os fagócitos e células apoptóticas em embriões inteiros devido à sua espessura. Para superar essa limitação, desenvolvemosUm novo teste de fagocitose em embriões de Drosophila . Em nosso método, para conter facilmente os hemocitros fagocitantes, os embriões integrais são homogeneizados para preparar células embrionárias dispersas. Os fagócitos são detectados pela imunocoloração de um marcador de fagócitos e as células apoptóticas são detectadas por TUNEL com estas células embrionárias dispersas. O uso de células embrionárias dispersas nos permite medir níveis de fagocitose in vivo como se realizássemos um teste de fagocitose in vitro que quantificasse precisamente o nível de fagocitose. Todos os genótipos de moscas podem ser empregados neste ensaio se desenvolverem o estágio 16 de embriões 20 , o estágio de desenvolvimento no qual a depuração celular apoptótica por fagocitose é a mais abundante. Este método tem a vantagem de avaliar quantitativamente o nível de fagocitose e, portanto, pode contribuir para a identificação de novas moléculas envolvidas na fagocitose de células apoptóticas in vivo .

<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

Os mecanismos moleculares subjacentes à fagocitose das células apoptóticas precisam ser esclarecidos com mais detalhes devido ao seu papel nas doenças imunes e inflamatórias intratáveis. Aqui desenvolvemos um método experimental para investigar a fagocitose quantitativamente usando a Drosophila da mosca da fruta, na qual a rede de genes que controla as reações de engulfment é conservada evolutivamente de mamíferos. A fim de detectar com precisão e contar fagócitos envolventes e não envolventes utilizando animais inteiros, os embriões de Drosophila foram homogeneizados para obter células dispersas, incluindo fagócitos e células apoptóticas. O uso de células embrionárias dispersas nos permite medir níveis de fagocitose in vivo como se realizássemos um teste de fagocitose in vitro em que seja possível observar todos os fagócitos e células apoptóticas em embriões inteiros e quantificar com precisão o nível de fagocitose. Confirmamos que este método reproduz os de estudos préviosQue identificou os genes necessários para a fagocitose das células apoptóticas. Este método permite analisar a absorção de células mortas e, quando combinado com a poderosa genética da Drosophila , revelará as complexas reações fagocíticas que compõem a migração, reconhecimento, engolfamento e degradação de células apoptóticas por fagócitos.

Para examinar a fagocitose das células apoptóticas, os embriões de Drosophila do estádio de desenvolvimento 16 foram coletados e preparados como células dispersas. Hemocitus, macrófagos de Drosophila , foram corados por imunocitoquímica para o marcador de hemicíticos &quot;Croquemort&quot; 17 , 22 usando um anticorpo específico 19 , 21 e as cél...

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

Aqui descrevemos um ensaio de fagocitose usando as células embrionárias dispersas de Drosophila . Ele nos permite quantificar de forma fácil e precisa os níveis de fagocitose in vivo e identificar novas moléculas necessárias para a fagocitose das células apoptóticas.

Ensaio de fagocitose para células apoptóticas em Drosophila Embriões

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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