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

Estamos agradecidos a Kaz Nagaosa y Akiko Shiratsuchi por su consejo.

1. Preparación <ol><li> Preparación de placas de agar de jugo de uva fresca <ol><li> Añadir 100 ml de agua a 4,4 g de agar y calentar la mezcla en un horno de microondas para disolver el agar. </li><li> Añadir 80 ml de jugo de uva fresca, 5 ml de ácido acético y 5 ml de etanol en solución de agar. </li><li> Verter aproximadamente 1,5 ml de la solución con una pipeta en cada portaobjetos de vidrio y dejar que se solidifique. </li></ol></li><li> Preparación de placas de 6 cm de placa de...

<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> En este documento se describió un ensayo de fagocitosis utilizando embriones de Drosophila . Mediante el uso de células embrionarias dispersas para medir la fagocitosis cuantitativamente, los hemocitos, los fagocitos profesionales de Drosophila , son inmunocorados para el marcador de hemocitos Croquemort o srpHemo- dirigido GFP, y las células apoptóticas son detectadas por TUNEL en este protocolo. El nivel de fagocitosis se expresa como un índice fagocítico contando el número tota...

En animales metazoarios, por ejemplo el nematodo Caenorhabditis elegans , la mosca de la fruta Drosophila melanogaster , y ratones y humanos, un gran número de células sufren apoptosis durante el desarrollo para modelar sus cuerpos y en la edad adulta para mantener la homeostasis 1 , 2 . Las células apoptóticas necesitan ser eliminadas rápidamente porque inducen inflamación en los tejidos circundantes liberando materiales intracelulares inmunogénicos si no se eliminan por completo 3 . Para facilitar la remoción rápida, las células apoptóticas presentan las denominadas señales de comer-me que son reconocidas por los receptores de engulfment de los fagocitos, y son eliminadas por fagocitosis 3 , 4 , 5 , 6 . Así, la fagocitosis desempeña un papel crucial en el mantenimiento de la homeostasis del huésped, y por lo tanto, elucidar la molécula<html> Los mecanismos subyacentes a la fagocitosis de las células apoptóticas son importantes. Los mecanismos responsables de la fagocitosis de las células apoptóticas parecen ser evolutivamente conservadas entre las especies en los nematodos, moscas y ratones [ 7] . Actualmente se dispone de varios ensayos de fagocitosis para evaluar la absorción de células apoptóticas en estos animales modelo. En C. elegans , 131 células somáticas sufren muerte celular programada durante el desarrollo, y los cadáveres de células son phagocytosed por las células vecinas, que son no fagocitos profesionales [ 8] . Por lo tanto, contar el número de cadáveres de células restantes en C. elegans indica el nivel de fagocitosis in vivo . En la búsqueda de mutantes de nematodos que muestran un mayor número de células muertas, varios genes necesarios para la fagocitosis han sido identificados y caracterizados genéticamente 9 , 10 ,S = &quot;xref&quot;&gt; 11 , 12 . Los ensayos de fagocitosis ex vivo con fagocitos de cultivo primario, generalmente macrófagos, se usan a menudo en ratones. Las células apoptóticas se preparan usando líneas celulares tales como células Jurkat, y se mezclan con fagocitos primarios. Después de una incubación durante varias horas, se cuenta el número total de fagocitos y de fagocitos engullantes para evaluar el nivel de fagocitosis. Como una modificación sofisticada de este método, el grupo de Nagata desarrolló un ensayo de fagocitosis ex vivo con células que expresan un ICAD resistente a caspasa (inhibidor de la DNasa activada por caspasa), en el que las células apoptóticas no sufren fragmentación apoptótica del ADN, Las células son fagocitadas. Cuando estas células se usan como dianas apoptóticas en un ensayo de fagocitosis, sólo el ADN de las células apoptóticas engullidas está fragmentado y teñido por marcado de extremos dUTP mediados por TdT (TUNEL). Por lo tanto, el leveL de la absorción de células apoptóticas se mide por el recuento de señales TUNEL en una mezcla de fagocitos y células apoptóticas [ 13] . En D. melanogaster , los fagocitos profesionales denominados hemocitos, los macrófagos de Drosophila , son responsables de la fagocitosis de las células apoptóticas 14 , 15 . Además de los ensayos de fagocitosis in vitro con líneas celulares de cultivo, se dispone de ensayos de fagocitosis in vivo con embriones enteros de Drosophila . Los embriones de Drosophila son una poderosa herramienta para examinar el nivel de engrosamiento de células apoptóticas debido a que muchas células sufren apoptosis y son fagocitadas por hemocitos durante el desarrollo embrionario 14 , 15 , 16 . Un ejemplo de un ensayo de fagocitosis in vivo es el método desarrollado por el grupo de Franc. En su método, los hemocitos sonProtegido por la inmunotinción de peroxidasina, un marcador de hemocito, las células apoptóticas se tiñen utilizando el tinte nuclear, 7-amino actinomicina D en embriones enteros de Drosophila , y el número de células positivas dobles se cuenta como una señal de fagocitosis 17 . Otro ejemplo de un ensayo de fagocitosis en embriones se basa en el concepto de método de Nagata descrito anteriormente; Sin embargo, la fagocitosis in vivo se evalúa utilizando los embriones de dCAD ( Drosophila caspasa-DNasa activada) moscas mutantes [ 18 , 19] . Estos ensayos de fagocitosis in vivo son útiles para observar directamente la fagocitosis in situ . Sin embargo, las dificultades están asociadas con la exclusión de cualquier sesgo posible en el paso de contar células fagocitosas porque es difícil observar todos los fagocitos y células apoptóticas en embriones enteros debido a su grosor. Para superar esta limitación, desarrollamosUn nuevo ensayo de fagocitosis en embriones de Drosophila . En nuestro método, para poder contar fácilmente los hemocitos fagocitados, se homogeneizan embriones enteros para preparar células embrionarias dispersas. Los fagocitos son detectados por la inmunotinción de un marcador de fagocitos, y las células apoptóticas son detectadas por TUNEL con estas células embrionarias dispersas. El uso de células embrionarias dispersas nos permite medir los niveles de fagocitosis in vivo como si se realizara un ensayo de fagocitosis in vitro que cuantificara con precisión el nivel de fagocitosis. Todos los genotipos de moscas pueden emplearse en este ensayo si se desarrollan hasta la etapa 16 de los embriones 20 , la etapa de desarrollo en la que la depuración de las células apoptóticas por fagocitosis es la más abundante. Este método tiene la ventaja de evaluar cuantitativamente el nivel de fagocitosis y, por tanto, puede contribuir a la identificación de nuevas moléculas implicadas en la fagocitosis 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

Los mecanismos moleculares subyacentes a la fagocitosis de las células apoptóticas necesitan ser aclarados con más detalle debido a su papel en las enfermedades inmunes e inflamables intratables. En este documento se desarrolló un método experimental para investigar la fagocitosis cuantitativamente utilizando la mosca de la fruta Drosophila , en el que la red de genes que controla las reacciones de engulfment se conserva evolutivamente de los mamíferos. Con el fin de detectar con precisión y contando engullir y no engolfar los fagocitos con animales enteros, embriones de Drosophila se homogeneizaron para obtener células dispersadas incluyendo fagocitos y células apoptóticas. El uso de células embrionarias dispersas nos permite medir los niveles de fagocitosis in vivo como si se realizara un ensayo de fagocitosis in vitro en el que es posible observar todos los fagocitos y células apoptóticas en embriones enteros y cuantificar con precisión el nivel de fagocitosis. Se confirmó que este método reproduce los de estudios previosQue identificó los genes necesarios para la fagocitosis de las células apoptóticas. Este método permite analizar la absorción de células muertas y, cuando se combina con la poderosa genética de Drosophila , revelará las complejas reacciones fagocíticas que comprenden la migración, el reconocimiento, el engolfamiento y la degradación de las células apoptóticas por los fagocitos.

Con el fin de examinar la fagocitosis de las células apoptóticas, Drosophila embriones de desarrollo etapa 16 fueron recogidos y preparados como células dispersas. Los hemocitos, macrófagos de Drosophila , se tiñeron por inmunocitoquímica para el marcador de hemocito &quot;Croquemort&quot; 17 , 22 usando un anticuerpo específico 19 , 21 , y las c?...

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

En el presente documento se describe un ensayo de fagocitosis utilizando las células embrionarias dispersas de Drosophila . Nos permite cuantificar con facilidad y precisión los niveles de fagocitosis in vivo , e identificar nuevas moléculas necesarias para la fagocitosis de las células apoptóticas.

Ensayo de Fagocitosis para Células Apoptóticas en<em&gt; Drosophila</em&gt; Embriones

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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