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

Siamo grati a Kaz Nagaosa e Akiko Shiratsuchi per il loro consiglio.

1. Preparazione <ol><li> Preparazione di piatti di agar freschi di uva <ol><li> Aggiungere 100 ml di acqua a 4,4 g di agar e riscaldare la miscela in un forno a microonde per sciogliere l&#39;agar. </li><li> Aggiungere 80 ml di succo fresco di uva, 5 ml di acido acetico e 5 ml di etanolo alla soluzione agar. </li><li> Versare circa 1,5 ml della soluzione con una pipetta su ogni vetretta di vetro e lasciarlo solidificare. </li></ol></li><li> Preparazione di piastre da 6 cm di agarosio ...

<ol>
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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> Qui abbiamo descritto un test di fagocitosi usando embrioni di Drosophila . Utilizzando cellule embrionali disperse per misurare la fagocitosi quantitativamente, gli emociti, i fagociti professionali di Drosophila , sono immunociti per il marker Crohnemort o il GHP driven by SrHemo , e le cellule apoptotiche vengono rilevate da TUNEL in questo protocollo. Il livello di fagocitosi è espresso come indice fagocitico contando il numero totale di emociti e fegocitosi emociti. L&#39;...

Negli animali metazoi, ad es. Il nematode Caenorhabditis elegans , la mosca di frutta Drosophila melanogaster , e topi e umani, un gran numero di cellule subiscono apoptosi durante lo sviluppo per modellare i loro corpi e in età adulta per mantenere l&#39;omeostasi 1 , 2 . Le cellule apoptotiche devono essere rapidamente rimosse perché inducono l&#39;infiammazione nei tessuti circostanti liberando materiali intracellulari immunogenici se non completamente rimossi 3 . Al fine di facilitare la rimozione rapida, le cellule apoptotiche presentano i cosiddetti segnali alimentari riconosciuti dai recettori di engagement dei fagociti e vengono eliminati dalla fagocitosi 3 , 4 , 5 , 6 . Pertanto, la fagocitosi svolge un ruolo cruciale nel mantenimento dell&#39;omeostasi ospite e, dunque, illustra la molecola<html> Lar meccanismi alla base della fagocitosi delle cellule apoptotiche è di importanza. I meccanismi responsabili della fagocitosi delle cellule apoptotici sembrano evoluzionalmente conservati tra le specie nei nematodi, mosche e topi 7 . Attualmente sono disponibili diversi dosaggi di fagocitosi per valutare l&#39;ingrasso di cellule apoptotiche in questi animali modello. In C. elegans , 131 cellule somatiche subiscono una morte cellulare programmata durante lo sviluppo, ei cadaveri cellulari sono fagocitati dalle cellule vicine, che sono fagociti non professionali 8 . Quindi, contando il numero di restanti cellule di cellule in C. elegans indica il livello di fagocitosi in vivo . Cercando mutanti di nematodi che mostrano un numero crescente di cellule morte, sono stati identificati diversi geni richiesti per la fagocitosi e caratterizzati geneticamente da 9 , 10 ,S = &quot;xref&quot;&gt; 11 , 12 . I campioni di fagocitosi ex vivo con fagociti di coltura primaria, generalmente macrofagi, vengono spesso utilizzati nei topi. Le cellule apoptotiche vengono preparate utilizzando linee cellulari come le cellule Jurkat e sono mescolate con fagociti primari. Dopo un&#39;incubazione per diverse ore, il numero totale di fagociti e fagociti inghiottiti vengono contati per valutare il livello della fagocitosi. Come una sofisticata modifica di questo metodo, il gruppo di Nagata ha sviluppato un test di fagocitosi ex vivo con le cellule che esprimono un ICAD resistente alla caspasi (inibitore della DNasi attivata da caspasi), in cui le cellule apoptotiche non subiscono la frammentazione del DNA apoptotica, ma il DNA è ancora ceduto quando Le cellule sono fagocitose. Quando queste cellule vengono utilizzate come obiettivi apoptotici in un dosaggio di fagocitosi, solo il DNA delle cellule apoptotiche inghiottite viene frammentato e macchiato mediante etichettatura TUTT-mediata di dUTP (TUNEL). Pertanto, la levaL di ingrasso cellulare apoptotico è misurato contando i segnali TUNEL in una miscela di fagociti e di cellule apoptotiche 13 . In D. melanogaster , fagociti professionali chiamati emociti, macrofagi Drosophila , sono responsabili della fagocitosi delle cellule apoptotiche 14 , 15 . Oltre ai dosaggi di fagocitosi in vitro con linee di cellule di coltura, sono disponibili test di fagocitosi in vivo con tutti gli embrioni Drosophila . Gli embrioni di Drosophila sono un potente strumento per esaminare il livello di induzione di cellule apoptotiche perché molte cellule subiscono l&#39;apoptosi e sono fagocitate da emociti durante lo sviluppo embrionale 14 , 15 , 16 . Un esempio di un test di fagocitosi in vivo è il metodo sviluppato dal gruppo di Franc. Nel loro metodo, gli emociti sono deTossicodipendente perossidasina, un marker emocitico, le cellule apoptotiche vengono colorate utilizzando il colorante nucleare, 7-amino actinomicina D negli embrioni interi Drosophila e il numero di cellule doppie positive viene contato come segnale di fagocitosi 17 . Un altro esempio di un test di fagocitosi sugli embrioni si basa sul concetto del metodo di Nagata descritto in precedenza; tuttavia, fagocitosi in vivo è valutata utilizzando gli embrioni di DCAD (Drosophila caspasi-attivato DNasi) mutante vola 18, 19. Questi test di fagocitosi in vivo sono utili per osservare direttamente la fagocitosi in situ . Tuttavia, le difficoltà sono associate ad escludere ogni possibile polarizzazione nella fase di conteggio delle cellule fagocitose perché è difficile osservare tutti i fagociti e le cellule apoptotiche in embrioni interi a causa dello spessore. Al fine di superare questa limitazione, ci sviluppiamoEd un nuovo dosaggio di fagocitosi negli embrioni di Drosophila . Nel nostro metodo, per poter facilmente contare gli emociti fagocitosi, tutti gli embrioni vengono omogeneizzati per preparare cellule embrionali disperse. I fagociti sono rilevati mediante l&#39;immunostaining di un marker di fagocita e le cellule apoptotiche sono rilevate da TUNEL con queste cellule embrionali disperse. L&#39;uso di cellule embrionali disperse ci permette di misurare i livelli di fagocitosi in vivo come se abbiamo effettuato un test di fagocitosi in vitro che precisamente quantifica il livello della fagocitosi. Tutti i genotipi di mosche possono essere impiegati in questo saggio se si sviluppano allo stadio 16 degli embrioni 20 , lo stadio di sviluppo in cui la clearance delle cellule apoptotiche mediante la fagocitosi è la più abbondante. Questo metodo ha il vantaggio di valutare quantitativamente il livello della fagocitosi e, quindi, può contribuire all&#39;identificazione di nuove molecole coinvolte nella fagocitosi delle cellule apoptotiche 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

I meccanismi molecolari alla base della fagocitosi delle cellule apoptotici devono essere chiariti in modo più dettagliato a causa del suo ruolo in malattie intractable e immunitarie e infiammatorie. Noi qui abbiamo sviluppato un metodo sperimentale per studiare la fagocitosi in modo quantitativo utilizzando la mosca Drosophila di frutta, in cui la rete genica che controlla le reazioni di engulfment è evolutamente conservata dai mammiferi. Al fine di rilevare e contare con precisione i fagociti inghiottiti e inattivi con animali interi, gli embrioni di Drosophila sono stati omogeneizzati per ottenere le cellule disperse, compresi i fagociti e le cellule apoptotiche. L&#39;uso di cellule embrionali disperse ci permette di misurare i livelli di fagocitosi in vivo come se avessimo eseguito un test di fagocitosi in vitro in cui è possibile osservare tutti i fagociti e le cellule apoptotiche in embrioni interi e precisamente quantificare il livello di fagocitosi. Abbiamo confermato che questo metodo riproduce quelli di studi precedentiChe ha identificato i geni richiesti per la fagocitosi delle cellule apoptotiche. Questo metodo permette di analizzare l&#39;engulfment di cellule morte e, in combinazione con la potente genetica di Drosophila , rivelerà le complesse reazioni fagocitiche che comprendono la migrazione, il riconoscimento, l&#39;engulfment e il degrado delle cellule apoptotiche mediante fagociti.

Al fine di esaminare la fagocitosi delle cellule apoptotiche, gli embrioni Drosophila dello stadio di sviluppo sono stati raccolti e preparati come cellule disperse. Gli emociti, i macrofagi Drosophila , sono stati macchiati mediante immunocitochimica per il marcatore emocitario &quot;Croquemort&quot; 17 , 22 utilizzando un anticorpo specifico 19 , 21 e ...

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

Qui descriviamo un test di fagocitosi usando le cellule embrionali disperse di Drosophila . Ci permette di quantificare con facilità e precisione i livelli di fagocitosi in vivo e di identificare nuove molecole necessarie per la fagocitosi delle cellule apoptotiche.

Analisi della fagocitosi per le cellule apoptotiche in<em&gt; Drosophila</em&gt; Embrioni

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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