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

Vi er taknemmelige for Kaz Nagaosa og Akiko Shiratsuchi for deres råd.

1. Fremstilling <ol><li> Fremstilling af friske druesaft-agarplader <ol><li> Tilsæt 100 ml vand til 4,4 g agar og opvarm blandingen i en mikrobølgeovn for at opløse agar. </li><li> Tilsæt 80 ml frisk druesaft, 5 ml eddikesyre og 5 ml ethanol til agaropløsning. </li><li> Hæld ca. 1,5 ml af opløsningen med en pipette på hvert glasskinne og lad det størkne. </li></ol></li><li> Fremstilling af 6 cm skål agaroseplader <ol><li> Tilsæt 50 ml vand til 0,5 g agarose og opvarm blandi...

<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. 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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> Vi heri beskrev et fagocytose assay ved anvendelse af Drosophila embryoner. Ved at anvende dispergerede embryonale celler til måling af fagocytose kvantitativt, hæmocytter, Drosophila faglige fagocytter immunostained for hemocytmarkøren Croquemort eller srpHemo- driven GFP, og apoptotiske celler påvises af TUNEL i denne protokol. Niveauet af fagocytose udtrykkes som et fagocytisk indeks ved at tælle det totale antal hæmocytter og fagocytoserende hæmocytter. Anvendelsen af ​​di...

I metazoan dyr, f.eks. Nematoden Caenorhabditis elegans , frugtflyve Drosophila melanogaster og mus og mennesker, gennemgår et stort antal celler apoptose under udvikling for at forme deres kroppe og i voksenalderen for at opretholde homeostase 1 , 2 . Apoptotiske celler skal fjernes hurtigt, fordi de inducerer inflammation i det omgivende væv ved at frigive immunogene intracellulære materialer, hvis de ikke fjernes fuldstændigt 3 . For at lette hurtig fjernelse frembyder apoptotiske celler såkaldte eat-me-signaler, som genkendes af engulferingsreceptorerne af fagocytter, og elimineres af phagocytose 3 , 4 , 5 , 6 . Fagocytose spiller således en afgørende rolle i vedligeholdelsen af ​​værtshomeostase og dermed elucidere molekylet<html> Lar mekanismer, der ligger til grund for fagocytose af apoptotiske celler, er vigtige. Mekanismerne ansvarlige for phagocytose af apoptotiske celler synes at være evolutionelt konserveret blandt arter i nematoder, fluer og mus 7 . Adskillige fagocytose-analyser er i øjeblikket tilgængelige for at vurdere opfangningen af ​​apoptotiske celler i disse modeldyr. I C. elegans undergår 131 somatiske celler programmeret celledød under udvikling, og cellekroppe er fagocytosed af naboceller, som er ikke-faglige fagocytter 8 . Således tæller antallet af resterende cellekroppe i C. elegans angiver niveauet af fagocytose in vivo . Ved at søge efter nematode mutanter, der viser et forøget antal døde celler, er der blevet identificeret flere gener for phagocytose og genetisk karakteriseret 9 , 10 ,S = &quot;xref&quot;&gt; 11 , 12 . Eks vivo fagocytose assays med primære kultur fagocytter, generelt makrofager, anvendes ofte i mus. Apoptotiske celler fremstilles under anvendelse af cellelinjer, såsom Jurkat-celler, og blandes med primære fagocytter. Efter en inkubation i flere timer tælles det totale antal fagocytter og engulfing fagocytter for at vurdere niveauet af fagocytose. Som en sofistikeret modifikation af denne metode udviklede Nagatas gruppe et eks vivo fagocytose assay med celler, der udtrykker en caspase-resistent ICAD (inhibitor af caspaseaktiveret DNase), hvor apoptotiske celler ikke undergår apoptotisk DNA-fragmentering, men DNA spaltes stadig når Celler er phagocytosed. Når disse celler anvendes som apoptotiske mål i et fagocytose assay, fragmenteres kun DNA&#39;et af engulferede apoptotiske celler og farves med TdT-medieret DUTP nick end-mærkning (TUNEL). Derfor lever deL af apoptotisk celle-engulfering måles ved at tælle TUNEL-signaler i en blanding af phagocytter og apoptotiske celler 13 . I D. melanogaster er faglige fagocytter, der hedder hæmocytter, Drosophila- makrofager, ansvarlige for fagocytosen af ​​apoptotiske celler 14 , 15 . Ud over in vitro fagocytose-assays med kulturcellelinier er in vivo fagocytose-analyser med hele Drosophila- embryoner tilgængelige. Drosophila embryoner er et kraftfuldt redskab til at undersøge niveauet af apoptotisk celleopfyldning, fordi mange celler gennemgår apoptose og fagocytteres af hæmocytter under embryonisk udvikling 14 , 15 , 16 . Et eksempel på et in vivo fagocytose assay er metoden udviklet af Francs gruppe. I deres metode er hæmocytter deBeskadiget ved immunostaining af peroxidasin, en hæmocytmarkør, farves apoptotiske celler under anvendelse af det nukleare farvestof, 7-amino-actinomycin D i hele Drosophila- embryoner, og antallet af dobbelt positive celler tælles som et signal af fagocytose 17 . Et andet eksempel på et fagocytose-assay på embryoner er baseret på begrebet Nagatas metode beskrevet ovenfor; Imidlertid evalueres fagocytose in vivo under anvendelse af embryonerne af dCAD ( Drosophila caspase-aktiverede DNase) mutant fluer 18 , 19 . Disse in vivo fagocytose-assays er nyttige til direkte observation af fagocytose in situ . Vanskeligheder er imidlertid forbundet med at udelukke enhver mulig forspænding i trinnet med at tælle fagocytoseceller, fordi det er svært at observere alle fagocytter og apoptotiske celler i hele embryoner på grund af dens tykkelse. For at overvinde denne begrænsning udvikler viUdarbejdet et nyt fagocytose assay i Drosophila embryoner. I vores metode homogeniseres hele embryoner for at fremstille dispergerede embryonale celler for nemt at tælle fagocytoserende hæmocytter. Phagocytter detekteres ved immunforaining af en fagocytmarkør, og apoptotiske celler påvises af TUNEL med disse dispergerede embryonale celler. Anvendelsen af ​​dispergerede embryonale celler gør det muligt for os at måle in vivo fagocytose niveauer som om vi udførte et in vitro fagocytose assay, der præcist kvantificerer niveauet af fagocytose. Alle genotyper af fluer kan anvendes i dette assay, hvis de udvikler sig til stadium 16 af embryoner 20 , udviklingsstadiet, hvor apoptotisk celleklaration ved phagocytose er den mest rigelige. Denne metode har den fordel at kvantificere niveauet af phagocytose kvantitativt og kan således bidrage til identifikationen af ​​nye molekyler involveret i phagocytose af apoptotiske celler 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

De molekylære mekanismer, der ligger til grund for phagocytosen af ​​apoptotiske celler, skal præciseres mere detaljeret på grund af dets rolle i immune og inflammatoriske, uoprettelige sygdomme. Her har vi udviklet en eksperimentel metode til undersøgelse af fagocytose kvantitativt ved anvendelse af frugtflyve Drosophila , hvor gennetværket , der styrer engulferingsreaktioner, evolutionelt konserveres fra pattedyr. For at nøjagtigt detektere og tælle engulfing og un-engulfing fagocytter under anvendelse af hele dyr, blev Drosophila embryoner homogeniseret for at opnå dispergerede celler, herunder phagocytter og apoptotiske celler. Anvendelsen af ​​dispergerede embryonale celler gør det muligt for os at måle in vivo fagocytose niveauer som om vi udførte et in vitro fagocytose assay, hvor det er muligt at observere alle fagocytter og apoptotiske celler i hele embryoner og præcist kvantificere niveauet af fagocytose. Vi bekræftede, at denne metode gengiver de tidligere studierDer identificerede de gener, der kræves til fagocytose af apoptotiske celler. Denne metode gør det muligt at analysere opbrydning af døde celler, og når de kombineres med Drosophila &#39;s kraftige genetik, vil de afsløre de komplekse fagocytiske reaktioner, der består af migration, genkendelse, opfangning og nedbrydning af apoptotiske celler af fagocytter.

For at undersøge phagocytosen af ​​apoptotiske celler blev Drosophila- embryoner fra udviklingsstadiet 16 opsamlet og fremstillet som dispergerede celler. Hæmocytter, Drosophila makrofager, blev farvet ved immuncytokemi for hæmocyt markør &quot;Croquemort&quot; 17, 22 under anvendelse af et specifikt antistof 19, 21, og apoptotiske celler blev fa...

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

Heri beskrives et fagocytose assay under anvendelse af de dispergerede embryonale celler af Drosophila . Det sætter os i stand til nemt og præcist at kvantificere phagocytose niveauer in vivo og at identificere nye molekyler, der er nødvendige for phagocytose af apoptotiske celler.

Phagocytosis Assay for Apoptotiske Celler i<em&gt; Drosophila</em&gt; Embryoer

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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