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 to 0.5 g of agarose, and heat the mixture in a microwave oven to dissolve agarose.</li>
		<li>Pour approximately 2.5 mL of agarose solution onto a 6 cm dish with a pipette.</li>
	</ol>
	</li>
</ol>
2. Stage 16 Embryo Collection
<ol>
	<li>Collect adult flies, and add 200 females and 200 males into a 50 mL conical tube.</li>
	<li>Put a plate of fresh grape juice agar on yeast into the 50-mL tube, and close with a sponge cap.</li>
	<li>Incubate the flies at 16 &#176;C in the light for 2 - 3 days. Change the plate once a day.</li>
	<li>Move the flies to the dark at 25 &#176;C for 1 h.</li>
	<li>Change the old plate to a new one without yeast. Allow flies to lay eggs for 2 h.</li>
	<li>Collect the plate and incubate at 16 &#176;C for 26 h.</li>
	<li>Collect embryos from the plate with a paint brush into 1 mL of PBS containing 0.2% (v/v) Triton X-100.</li>
	<li>Wash embryos twice with 1 mL of PBS containing 0.2% (v/v) Triton X-100.</li>
	<li>In order to remove the chorion, add 1.2 mL of sodium hypochlorite solution (2.2 - 3.4% Cl) to embryos for 3 min.</li>
	<li>Wash embryos 4 times with 1 mL of PBS containing 0.2% (v/v) Triton X-100.</li>
	<li>Place embryos on 6-cm dish agarose plates. Pick up stage 16 embryos under a microscope as described by Roberts20. Collect approximately 50 embryos in a 1.5-mL Treff micro test tube with a micropipette.</li>
</ol>
3. Preparation of Embryonic Cells
<ol>
	<li>Wash embryos twice with 150 &#181;L of PBS.</li>
	<li>Homogenize embryos 30 times in a 1.5 mL micro test tube and pellet mixer in the presence of 200 &#181;L of collagenase (0.25% (w/v)) in PBS.</li>
	<li>Disperse embryonic cells by pipetting 10 times, and move the cell suspension to a 1.5 mL tube. Incubate cells at 37 &#176;C for 1 min in a water bath. Repeat this step twice.</li>
	<li>Add 800 &#181;L of PBS to the cell suspension, and centrifuge at 1400 x g at 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant and add 200 &#181;L of trypsin (0.25% (w/v)) in PBS to the cells. Disperse embryonic cells by pipetting 50 times.</li>
	<li>Filtrate the cell suspension with a 70 &#181;m Cell Strainer.</li>
	<li>In order to stop trypsin activity, add 40 &#181;L heat-inactivated fetal bovine serum to the filtrated cell suspension. Add 800 &#181;L of PBS and centrifuge at 1,400 x g at 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant, suspend precipitated cells in 200 &#181;L of PBS, and centrifuge at 1400 x g at 4 &#176;C for 5 min.</li>
	<li>Remove the supernatant, and suspend precipitated cells in 30 &#181;L of PBS.</li>
	<li>Mount the prepared cell suspension on aminopropyl triethoxysilane-coated glass slides, and wait for 10 - 15 min until cells attach to the glass slides.</li>
	<li>Remove the remaining solution on the glass slides with a pipette. Mount 60 - 70 &#181;L of PBS containing 4% (w/v) PFA (paraformaldehyde) onto cells for 10 - 15 min for fixation.</li>
	<li>Remove the fixation solution, and place glass slides into PBS for more than 1 min.</li>
</ol>
4. Staining of Hemocytes
<ol>
	<li>Immunostaining with an anti-Croquemort antibody
	<ol>
		<li>Serially soak glass slides in methanol for 10 min, in PBS containing 0.2% (v/v) Triton X-100 for 10 min, and then in PBS for 10 min.</li>
		<li>Mount 20 &#181;L of 5% (v/v) whole swine serum in PBS containing 0.2% (v/v) Triton X-100 on cells for blocking. Incubate slides at room temperature for 20 min.</li>
		<li>Remove the solution from glass slides, and mount 20 &#181;L of anti-Croquemort antiserum19,21 (0.1% (v/v)) in PBS containing 0.2% (v/v) Triton X-100 on cells. Incubate slides at 4 &#176;C overnight.</li>
		<li>Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.</li>
		<li>Soak glass slides in PBS for 10 min.</li>
		<li>Mount 20 &#181;L of alkaline phosphatase-labeled anti-rat IgG (0.5% (v/v)) in PBS containing 0.2% (v/v) Triton X-100 and 5% (v/v) whole swine serum on cells at room temperature for 1 h.</li>
		<li>Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.</li>
		<li>Soak glass slides in buffer containing 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2 for 10 min.</li>
		<li>Mount phosphatase substrate solution containing 0.23 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (BCIP), 0.35 mg/mL nitro blue tetrazolium (NBT), 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2 on cells.</li>
		<li>Observe cells under a microscope for proper staining. When strong purple signals appear in the granules of hemocytes, remove the substrate solution, and soak glass slides in buffer consisting of 10 mM Tris-HCl, pH 8.5 and 1 mM EDTA. Staining for approximately 5 - 10 min is recommended.</li>
	</ol>
	</li>
	<li>Immunostaining with an anti-GFP antibody (another option for the staining of hemocytes)
	<ol>
		<li>Serially soak glass slides in methanol for 10 min, PBS containing 0.2% (v/v) Triton X-100 for 10 min, and PBS for 10 min.</li>
		<li>Mount 20 &#181;L of 5% (v/v) whole swine serum in PBS containing 0.2% (v/v) Triton X-100 on cells for blocking. Incubate slides at room temperature for 20 min.</li>
		<li>Remove the solution on glass slides, and mount 20 &#181;L of the mouse anti-GFP antibody (1% (v/v))/PBS containing 0.2% (v/v) Triton X-100 on cells. Incubate slides at room temperature overnight.</li>
		<li>Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.</li>
		<li>Soak glass slides in PBS for 10 min.</li>
		<li>Mount 20 &#181;L of alkaline phosphatase-labeled anti-mouse IgG (0.5% (v/v)) in PBS containing 0.2% (v/v) Triton X-100 and 5% (v/v) whole swine serum on cells at room temperature for 1 h.</li>
		<li>Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.</li>
		<li>Soak glass slides in buffer consisting of 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2 for 10 min.</li>
		<li>Mount the phosphatase substrate solution containing 0.23 mg/mL BCIP, 0.35 mg/mL NBT, 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2 on cells.</li>
		<li>Observe cells under a microscope for proper staining. When purple signals appear in whole hemocytes, remove the substrate solution and soak slides in buffer consisting of 10 mM Tris-HCl, pH8.5 and 1 mM EDTA. Staining for approximately 10 - 15 min is recommended.</li>
	</ol>
	</li>
</ol>
5. Stain Apoptotic Cells by TUNEL
<ol>
	<li>Soak glass slides in PBS for 5 min.</li>
	<li>Repeat with new PBS.</li>
	<li>Mount Equilibration buffer (See Materials Table) on cells for 10 min.</li>
	<li>Remove the solution from glass slides, and mount 20 &#181;L of TdT solution containing 5 &#181;L of terminal deoxynucleotidyl transferase and 15 &#181;L of Reaction Buffer on cells at 37 &#176;C for 1 h.</li>
	<li>Remove the solution from glass slides, and soak slides in buffer consisting of 0.5 mL STOP/Wash buffer and 17 mL of water.</li>
	<li>Soak slides in PBS for 5 min 3 times.</li>
	<li>Mount 20 &#181;L of Anti-Digoxigenin-Peroxidase on cells at room temperature for 30 min.</li>
	<li>Soak glass slides in PBS for 5 min 4 times.</li>
	<li>Soak glass slides in peroxidase substrate solution consisting of 30 mL of 50 mM Tris-HCl, pH7.5 containing a full micro spatula of 3,3&#39;-diaminobenzidine tetrahydrichloride (DAB), and 0.002% (v/v) H2O2 for 30 s.</li>
	<li>Repeat step 5.9 until apoptotic cells are stained brown. Soak glass slides in water to stop the peroxidase reaction.</li>
	<li>Enclose cells with water for observation.</li>
</ol>
6. Measure the Level of Phagocytosis of Apoptotic Cells
<ol>
	<li>Observe samples under a light microscope in order to assess the level of phagocytosis. Count Croquemort-positive cells or GFP-positive cells as hemocytes, TUNEL-positive cells as apoptotic cells, and double positive cells as phagocytosing hemocytes.</li>
	<li>The phagocytic index is defined as the number of double positive cells to the total number of Croquemort-positive cells or GFP-positive cells. It is recommended that more than 300 hemocytes are observed.</li>
</ol>

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The authors declare that they have no competing financial interests.

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 enables us to observe all phagocytes and apoptotic cells in whole embryos and to precisely quantify the level of phagocytosis. In addition, our method detects hemocytes and apoptotic cells with dyes that are observable under a light microscope, not a fluorescence microscope. This facilitates the assessment of phagocytosis levels because it is possible to observe hemocytes and apoptotic cells simultaneously without changing any filter.
The following points are critical for staining embryonic cells. In the fixation step of embryonic cells, fresh PFA prepared within two weeks needs to be used. In the immunostaining of Croquemort, the incubation of cells with anti-Croquemort needs to be performed at 4 &#176;C. In TUNEL staining, the incubation of cells with DAB needs to be performed by carefully checking under a microscope to avoid excessive staining. TUNEL visualizes fragmented DNA by detecting 3&#39;-OH, which is abundant in apoptotic cells. However, normal cells also have 3&#39;-OH in the 3&#39;-terminal of DNA, but at a lower level than that in apoptotic cells. Therefore, an incubation of cells with DAB for too long will stain not only apoptotic cells, but also normal cells. The observation of cells every 15 or 30 s during DAB staining is recommended to prevent this.
In this protocol, although quantification of the level of phagocytosis is fast and precise, some information such as the site of engulfment or distribution of hemocytes is lost. We previously showed that this assay and an in situ phagocytosis assay using whole embryos produced similar results in terms of the phagocytic index21. Therefore, we recommend performing an in situ phagocytosis assay in parallel in order to accurately interpret the results obtained in some cases.
Drosophila have another professional phagocyte, glial cells, in the central nervous system. Although we do not describe how to assess the phagocytosis of apoptotic neurons by glial cells in embryos in this protocol, the same protocol is applied to quantify the level of engulfment as that reported by Kuraishi et al., EMBO.J21. Likewise, this assay suggests that TUNEL positive cells in Drosophila embryos are likely to be phagocytosed by non-professional phagocytes. Phagocytosis by those unidentified phagocytes with unknown engulfment receptors might explain the result (Figure 1D) that the total number of apoptotic cells in embryonic cells seems not to be increased in a phagocytosis gene mutant.
In conclusion, this protocol has successfully led to the discovery of genes required for the phagocytosis of apoptotic cells by professional phagocytes.24,25,28. Based on the evolutionary conservation of the molecular mechanisms underlying phagocytosis7, revealing gene functions in Drosophila embryos that involve complex phagocytosis reactions may provide meaningful insights into the phagocytic clearance of dead cells, which is related to inflammatory disorders and autoimmune diseases in mammals29.

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 embryonic cells (Figure 1A). Croquemort, a Drosophila CD36-related receptor, is specifically expressed on all embryonic hemocytes22, and has been shown genetically to be involved in the clearance of apoptotic cells in Drosophila embryos17. Croquemort-positive cells have purple signals in the small granules of their cells. TUNEL-positive cells show a brown signal in whole corpuses. TUNEL-positive cells are smaller than other cells, and some are inside Croquemort-positive cells, which are considered to be phagocytosed dead cells. Between 2 and 10% Croquemort-positive cells and 1 - 5% TUNEL-positive cells are normally observed in all embryonic cells.
In Drosophila, there are two signaling pathways for the engulfment of apoptotic cells. Two phagocytosis receptors for the corresponding pathways, Draper and integrin &#945;PS3&#946;&#957;, independently recognize dead cells19,21,23,24,25. Draper is a transmembrane protein which possesses atypical epidermal growth factor (EGF)-like sequences in the extracellular region and the two phosphorylatable motifs NPxY and YxxL in the intracellular portion26. Integrin &#945;PS3&#946;&#957; is also a transmembrane receptor consisting of a heterodimer of &#945; and &#946; subunits25. Figure 1B shows the ratio of phagocytosing hemocytes to total hemocytes in wild-type or drpr or Itgbn single mutant embryos. By counting all Croquemort-positive cells (total hemocytes) and Croquemort- and TUNEL-double positive cells (phagocytosing hemocytes), we calculated the phagocytic index as the number of phagocytosing hemocytes to the total number of hemocytes. The phagocytic index was lower in the drpr or Itgbn mutant than in the wild type, while the numbers of hemocytes and apoptotic cells were similar (Figure 1C-D), indicating that these two genes are required for apoptotic cell engulfment, as previously reported.
When an anti-Croquemort antibody is not available or mutant lines with altered Croquemort expression are examined, we need to select another option to detect hemocytes. Embryos with a GAL4 driver controlled by a hemocyte-specific promoter (srpHemo-GAL4), and the GFP gene controlled by an upstream activation sequence (UAS) including the GAL4-binding site (UAS-EGFP), have GFP-labeled hemocytes27, which enables us to detect hemocytes using anti-GFP. Figure 2A shows an image obtained after detecting hemocytes and apoptotic cells with immunostaining using an anti-GFP antibody, and TUNEL in embryonic cells with srpHemo-GAL4UAS-EGFP. While an anti-Croquemort antibody stains small granules in cells, an anti-GFP antibody stains whole hemocytes. Similar to Figure 1A, 2 - 6% GFP-positive cells and 1 - 5% TUNEL-positive cells are observed in all embryonic cells. Some TUNEL-positive cells are detected inside GFP-positive cells, which are considered to be phagocytosed dead cells. RNAi-mediated knockdown is easily applied to assess any genes for their involvement in phagocytosis by crossing flies with the UAS-dsRNA of the candidate genes with srpHemo-GAL4UAS-EGFP. The RNAi-mediated knockdown of drpr or Itgbn reduced the phagocytic index (Figure 2B), whereas the numbers of hemocytes and apoptotic cells in embryonic cells was comparable (Figure 2C-2D), indicating again that these phagocytosis receptors are involved in the phagocytosis of apoptotic cells. Similar phagocytic indexes are obtained from both hemocyte detection methods, suggesting that both are compatible.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56352/56352fig1.jpg" /> 
Figure 1: Staining of Embryonic Cells by an Anti-Croquemort Antibody and TUNEL. (A) Bright field images of dispersed embryonic cells from the w1118 strain by immunostaining with an anti-Croquemort antibody and TUNEL. Magnified views of representative positively or negatively stained cells (top 4 panels) and a low-power field (bottom panel) are shown. Scale bars, 5 &#181;m (top); 50 &#181;m (bottom). (B) The ratio of Croquemort-positive hemocytes with the TUNEL signal to all Croquemort-positive hemocytes in the drpr or Itgbn mutant was analyzed with dispersed embryonic cells. (C) The ratio of Croquemort-positive hemocytes to all cells in the drpr or Itgbn mutant was analyzed with dispersed embryonic cells. (D) The ratio of TUNEL-positive apoptotic cells to all cells in the drpr or Itgbn mutant was analyzed with dispersed embryonic cells. Genotypes; w1118(wild-type control), drpr&#916;5 (drpr mutant), and Itgbn2 (integrin &#946;&#957; mutant). Data were representative of three independent experiments and analyzed by the Student&#39;s t-test. Approximately 300 Croquemort-positive hemocytes were observed in each experiment, and values represent the mean&#177;S.D. of three microscopic fields. *; p &#60;0.05, n.s.; difference not significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56352/56352fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56352/56352fig2.jpg" /> 
Figure 2: Staining of Embryonic Cells by Anti-GFP and TUNEL. (A) Bright field images of dispersed embryonic cells from the srpHemo-GAL4UAS-EGFP/+ strain by immunostaining with an anti-GFP antibody and TUNEL. Magnified views of representative positively or negatively stained cells (top 4 panels) and a low-power field (bottom panel) are shown. Scale bars = 5 &#181;m (top); 50 &#181;m (bottom). (B) The ratio of GFP-positive hemocytes with the TUNEL signal to all GFP-positive hemocytes in RNAi-mediated knockdown flies of drpr or Itgbn was analyzed with dispersed embryonic cells. (C) The ratio of GFP-positive hemocytes to all cells in RNAi-mediated knockdown flies of drpr or Itgbn was analyzed with dispersed embryonic cells. (D) The ratio of TUNEL-positive apoptotic cells to all cells in RNAi-mediated knockdown flies of drpr or Itgbn was analyzed with dispersed embryonic cells. Genotypes; RNAi - : y w/w; srpHemo-GAL4 UAS-EGFP/+, RNAi drpr: y w/w; srpHemo-GAL4 UAS-EGFP/+; UAS-drpr-IR/+, RNAi Itgbn: y w/w; srpHemo-GAL4 UAS-EGFP/+; UAS-Itgbn-IR/+. Data were representative of three independent experiments and analyzed by the Student&#39;s t-test. Approximately 300 GFP-positive hemocytes were observed in each experiment, and values represent the mean&#177;S.D. of three microscopic fields. *; p &#60;0.05, n.s.; difference not significant. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56352/56352fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

phagocytosis-assay-for-apoptotic-cells-in-drosophila-embryos

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9.8K Views.  Kanazawa University. 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.

Video: Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled 
during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most 
effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by 
releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria 
or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, 
migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the 
infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold 
Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal 
defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the 
environment. Here, an ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an intricate network of inducers, detectors, and activators rapidly responds to neuron damage, infection or other immune infractions. This inflammation of immune cells is intimately associated with the pathology of neurodegenerative disorders, such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease and ALS. Under compromised disease states, chronic inflammation, intended to minimize neuron damage, may lead to an over-excitation of the immune cells, ultimately resulting in the exacerbation of disease progression. For example, loss of dopaminergic neurons in the midbrain, a hallmark of PD, is accelerated by the excessive activation of the inflammatory response. Though the cause of PD is largely unknown, exposure to environmental toxins has been implicated in the onset of sporadic cases. The herbicide paraquat, for example, has been shown to induce Parkinsonian-like pathology in several animal models, including Drosophila melanogaster. Here, we have used the conserved innate immune response in Drosophila to develop an assay capable of detecting varying levels of nitric oxide, a cell-signaling molecule critical to the activation of the inflammatory response cascade and targeted neuron death. Using paraquat-induced neuronal damage, we assess the impact of these immune insults on neuroinflammatory stimulation through the use of a novel, quantitative assay. Whole brains are fully extracted from flies either exposed to neurotoxins or of genotypes that elevate susceptibility to neurodegeneration then incubated in cell-culture media. Then, using the principles of the Griess reagent reaction, we are able to detect minor changes in the secretion of nitric oxide into cell-culture media, essentially creating a primary live-tissue model in a simple procedure. The utility of this model is amplified by the robust genetic and molecular complexity of Drosophila melanogaster, and this assay can be modified to be applicable to other Drosophila tissues or even other small, whole-organism inflammation models.

Novel Whole-tissue Quantitative Assay of Nitric Oxide Levels in Drosophila Neuroinflammatory Response

Levels of the inflammatory cell-signaling molecule nitric oxide (NO) are commonly assayed using Griess reagent. In this protocol, we have created a modified Griess assay utilizing live Drosophila brain tissue in order to detect the secretion of NO in a simple, quantifiable and highly repeatable method.

Neuroinflammation is a complex innate immune response vital to the healthy function of the central nervous system (CNS). Under normal conditions, an ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Alveolar Macrophage Phagocytosis and Bacteria Clearance in Mice

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the removal of dead cells or foreign particles, and in the resolution of inflammatory responses and tissue remodeling, processes that are mediated by various surface receptors of the AMs. Here, we report methods for the analysis of the phagocytic function of AMs using in vitro and in vivo assays and experimental strategies to differentiate between the pattern recognition receptor-, complement receptor-, and Fc gamma receptor-mediated phagocytosis. Finally, we discuss a method to establish and characterize a P. aeruginosa pneumonia model in mice to assess bacterial clearance in vivo. These assays represent the most common methods to evaluate AM functions and can also be used to study macrophage function and bacterial clearance in other organs.

Here we report common methods to analyze the phagocytic function of murine alveolar macrophages and bacterial clearance from the lung. These methods study in vitro phagocytosis of fluorescein isothiocyanate beads and in vivo phagocytosis of Pseudomonas aeruginosa Green Fluorescent Protein. We also describe a method for clearing P. aeruginosa in mice.

Alveolar macrophages (AMs) guard the alveolar space of the lung. Phagocytosis by AMs plays a critical role in the defense against invading pathogens, the ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

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