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

We are grateful to Kaz Nagaosa and Akiko Shiratsuchi for their advice.

1. Preparation
<ol>
	<li>Preparation of fresh grape juice agar plates
	<ol>
		<li>Add 100 mL of water to 4.4 g of agar, and heat the mixture in a microwave oven to dissolve agar.</li>
		<li>Add 80 mL of fresh grape juice, 5 mL of acetic acid, and 5 mL of ethanol to agar solution.</li>
		<li>Pour approximately 1.5 mL of the solution with a pipette onto each glass slide, and allow it to solidify.</li>
	</ol>
	</li>
	<li>Preparation of 6 cm dish agarose plates
	<ol>
		<li>Add 50 mL water ...

<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>

We herein described a phagocytosis assay using Drosophila embryos. By using dispersed embryonic cells to measure phagocytosis quantitatively, hemocytes, Drosophila professional phagocytes, are immunostained for the hemocyte marker Croquemort or srpHemo-driven GFP, and apoptotic cells are detected by TUNEL in this protocol. The level of phagocytosis is expressed as a phagocytic index by counting the total number of hemocytes and phagocytosing hemocytes. The use of dispersed embryonic cells enabl...

In metazoan animals, e.g. the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mice and humans, a large number of cells undergo apoptosis during development to shape their bodies and in adulthood to maintain homeostasis1,2. Apoptotic cells need to be rapidly removed because they induce inflammation in the surrounding tissues by releasing immunogenic intracellular materials if not completely removed3. In order to facilitate rapid removal, apoptotic cells present so-called eat-me signals that are recognized by the engulfment receptors of phagocytes, and are eliminated by phagocytosis3,4,5,6. Thus, phagocytosis plays a crucial role in the maintenance of host homeostasis, and hence, elucidating the molecular mechanisms underlying the phagocytosis of apoptotic cells is of importance.
The mechanisms responsible for the phagocytosis of apoptotic cells appear to be evolutionally conserved among species in the nematodes, flies, and mice7. Several phagocytosis assays are currently available to assess the engulfment of apoptotic cells in these model animals. In C. elegans, 131 somatic cells undergo programmed cell death during development, and cell corpses are phagocytosed by neighboring cells, which are non-professional phagocytes8. Thus, counting the number of remaining cell corpses in C. elegans indicates the level of phagocytosis in vivo. By searching for nematode mutants that show an increased number of dead cells, several genes required for phagocytosis have been identified and genetically characterized9,10,11,12.
Ex vivo phagocytosis assays with primary culture phagocytes, generally macrophages, are often utilized in mice. Apoptotic cells are prepared using cell lines such as Jurkat cells, and are mixed with primary phagocytes. After an incubation for several hours, the total numbers of phagocytes and engulfing phagocytes are counted in order to assess the level of phagocytosis. As a sophisticated modification of this method, Nagata&#39;s group developed an ex vivo phagocytosis assay with cells expressing a caspase-resistant ICAD (inhibitor of caspase-activated DNase), in which apoptotic cells do not undergo apoptotic DNA fragmentation, but DNA is still cleaved when cells are phagocytosed. When these cells are used as apoptotic targets in a phagocytosis assay, only the DNA of engulfed apoptotic cells is fragmented, and stained by TdT-mediated dUTP nick end labeling (TUNEL). Therefore, the level of apoptotic cell engulfment is measured by counting TUNEL signals in a mixture of phagocytes and apoptotic cells13.
In D. melanogaster, professional phagocytes named hemocytes, Drosophila macrophages, are responsible for the phagocytosis of apoptotic cells14,15. In addition to in vitro phagocytosis assays with culture cell lines, in vivo phagocytosis assays with whole Drosophila embryos are available. Drosophila embryos are a powerful tool for examining the level of apoptotic cell engulfment because many cells undergo apoptosis and are phagocytosed by hemocytes during embryonic development14,15,16. An example of an in vivo phagocytosis assay is the method developed by Franc&#39;s group. In their method, hemocytes are detected by the immunostaining of peroxidasin, a hemocyte marker, apoptotic cells are stained using the nuclear dye, 7-amino actinomycin D in whole Drosophila embryos, and the number of double positive cells is counted as a signal of phagocytosis17. Another example of a phagocytosis assay on embryos is based on the concept of Nagata&#39;s method described above; however, in vivo phagocytosis is evaluated using the embryos of dCAD (Drosophila caspase-activated DNase) mutant flies18,19. These in vivo phagocytosis assays are useful for directly observing phagocytosis in situ. However, difficulties are associated with excluding any possible bias in the step of counting phagocytosing cells because it is hard to observe all phagocytes and apoptotic cells in whole embryos due to its thickness.
In order to overcome this limitation, we developed a new phagocytosis assay in Drosophila embryos. In our method, in order to easily count phagocytosing hemocytes, whole embryos are homogenized to prepare dispersed embryonic cells. Phagocytes are detected by the immunostaining of a phagocyte marker, and apoptotic cells are detected by TUNEL with these dispersed embryonic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay that precisely quantifies the level of phagocytosis. All genotypes of flies may be employed in this assay if they develop to stage 16 of embryos20, the developmental stage at which apoptotic cell clearance by phagocytosis is the most abundant. This method has the advantage of assessing the level of phagocytosis quantitatively, and, thus, may contribute to the identification of new molecules involved in the phagocytosis of apoptotic cells 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

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and inflammatory intractable diseases. We herein developed an experimental method to investigate phagocytosis quantitatively using the fruit fly Drosophila, in which the gene network controlling engulfment reactions is evolutionally conserved from mammals. In order to accurately detect and count engulfing and un-engulfing phagocytes using whole animals, Drosophila embryos were homogenized to obtain dispersed cells including phagocytes and apoptotic cells. The use of dispersed embryonic cells enables us to measure in vivo phagocytosis levels as if we performed an in vitro phagocytosis assay in which it is possible to observe all phagocytes and apoptotic cells in whole embryos and precisely quantify the level of phagocytosis. We confirmed that this method reproduces those of previous studies that identified the genes required for the phagocytosis of apoptotic cells. This method allows the engulfment of dead cells to be analyzed, and when combined with the powerful genetics of Drosophila, will reveal the complex phagocytic reactions comprised of the migration, recognition, engulfment, and degradation of apoptotic cells by phagocytes.

In order to examine the phagocytosis of apoptotic cells, Drosophila embryos of developmental stage 16 were collected and prepared as dispersed cells. Hemocytes, Drosophila macrophages, were stained by immunocytochemistry for the hemocyte marker &#34;Croquemort&#34;17,22 using a specific antibody19,21, and apoptotic cells were stained by TUNEL in dispersed...

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

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

The molecular mechanisms underlying the phagocytosis of apoptotic cells need to be elucidated in more detail because of its role in immune and ...

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In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Isolation of Leukocytes from Human Breast Milk for Use in an Antibody-dependent Cellular Phagocytosis Assay of HIV Targets

Even in the absence of antiretroviral drugs, only ~15% of infants breastfed by HIV-infected mothers become infected, suggesting a strong protective effect ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...