Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 homeostasis^1,2. 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 phagocytosis^3,4,5,6. 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 characterized^9,10,11,12.

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'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 cells^14,15. 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 development^14,15,16. An example of an in vivo phagocytosis assay is the method developed by Franc'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's method described above; however, in vivo phagocytosis is evaluated using the embryos of dCAD (Drosophila caspase-activated DNase) mutant flies^18,19. 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.

Protokół

1. Preparation

1. Preparation of fresh grape juice agar plates
   1. Add 100 mL of water to 4.4 g of agar, and heat the mixture in a microwave oven to dissolve agar.
   2. Add 80 mL of fresh grape juice, 5 mL of acetic acid, and 5 mL of ethanol to agar solution.
   3. Pour approximately 1.5 mL of the solution with a pipette onto each glass slide, and allow it to solidify.
2. Preparation of 6 cm dish agarose plates
   1. Add 50 mL water to 0.5 g of agarose, and heat the mixture in a microwave oven to dissolve agarose.
   2. Pour approximately 2.5 mL of agarose solution onto a 6 cm dish with a pipette.

2. Stage 16 Embryo Collection

1. Collect adult flies, and add 200 females and 200 males into a 50 mL conical tube.
2. Put a plate of fresh grape juice agar on yeast into the 50-mL tube, and close with a sponge cap.
3. Incubate the flies at 16 °C in the light for 2 - 3 days. Change the plate once a day.
4. Move the flies to the dark at 25 °C for 1 h.
5. Change the old plate to a new one without yeast. Allow flies to lay eggs for 2 h.
6. Collect the plate and incubate at 16 °C for 26 h.
7. Collect embryos from the plate with a paint brush into 1 mL of PBS containing 0.2% (v/v) Triton X-100.
8. Wash embryos twice with 1 mL of PBS containing 0.2% (v/v) Triton X-100.
9. In order to remove the chorion, add 1.2 mL of sodium hypochlorite solution (2.2 - 3.4% Cl) to embryos for 3 min.
10. Wash embryos 4 times with 1 mL of PBS containing 0.2% (v/v) Triton X-100.
11. 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.

3. Preparation of Embryonic Cells

1. Wash embryos twice with 150 µL of PBS.
2. Homogenize embryos 30 times in a 1.5 mL micro test tube and pellet mixer in the presence of 200 µL of collagenase (0.25% (w/v)) in PBS.
3. Disperse embryonic cells by pipetting 10 times, and move the cell suspension to a 1.5 mL tube. Incubate cells at 37 °C for 1 min in a water bath. Repeat this step twice.
4. Add 800 µL of PBS to the cell suspension, and centrifuge at 1400 x g at 4 °C for 5 min.
5. Remove the supernatant and add 200 µL of trypsin (0.25% (w/v)) in PBS to the cells. Disperse embryonic cells by pipetting 50 times.
6. Filtrate the cell suspension with a 70 µm Cell Strainer.
7. In order to stop trypsin activity, add 40 µL heat-inactivated fetal bovine serum to the filtrated cell suspension. Add 800 µL of PBS and centrifuge at 1,400 x g at 4 °C for 5 min.
8. Remove the supernatant, suspend precipitated cells in 200 µL of PBS, and centrifuge at 1400 x g at 4 °C for 5 min.
9. Remove the supernatant, and suspend precipitated cells in 30 µL of PBS.
10. Mount the prepared cell suspension on aminopropyl triethoxysilane-coated glass slides, and wait for 10 - 15 min until cells attach to the glass slides.
11. Remove the remaining solution on the glass slides with a pipette. Mount 60 - 70 µL of PBS containing 4% (w/v) PFA (paraformaldehyde) onto cells for 10 - 15 min for fixation.
12. Remove the fixation solution, and place glass slides into PBS for more than 1 min.

4. Staining of Hemocytes

1. Immunostaining with an anti-Croquemort antibody
   1. 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.
   2. Mount 20 µL 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.
   3. Remove the solution from glass slides, and mount 20 µL of anti-Croquemort antiserum^19,21 (0.1% (v/v)) in PBS containing 0.2% (v/v) Triton X-100 on cells. Incubate slides at 4 °C overnight.
   4. Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.
   5. Soak glass slides in PBS for 10 min.
   6. Mount 20 µL 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.
   7. Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.
   8. Soak glass slides in buffer containing 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl₂ for 10 min.
   9. 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.
   10. 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.
2. Immunostaining with an anti-GFP antibody (another option for the staining of hemocytes)
   1. 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.
   2. Mount 20 µL 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.
   3. Remove the solution on glass slides, and mount 20 µL 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.
   4. Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.
   5. Soak glass slides in PBS for 10 min.
   6. Mount 20 µL 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.
   7. Soak glass slides in PBS containing 0.2% (v/v) Triton X-100 for 10 min 5 times.
   8. Soak glass slides in buffer consisting of 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl₂ for 10 min.
   9. 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.
   10. 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.

5. Stain Apoptotic Cells by TUNEL

1. Soak glass slides in PBS for 5 min.
2. Repeat with new PBS.
3. Mount Equilibration buffer (See Materials Table) on cells for 10 min.
4. Remove the solution from glass slides, and mount 20 µL of TdT solution containing 5 µL of terminal deoxynucleotidyl transferase and 15 µL of Reaction Buffer on cells at 37 °C for 1 h.
5. Remove the solution from glass slides, and soak slides in buffer consisting of 0.5 mL STOP/Wash buffer and 17 mL of water.
6. Soak slides in PBS for 5 min 3 times.
7. Mount 20 µL of Anti-Digoxigenin-Peroxidase on cells at room temperature for 30 min.
8. Soak glass slides in PBS for 5 min 4 times.
9. 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'-diaminobenzidine tetrahydrichloride (DAB), and 0.002% (v/v) H₂O₂ for 30 s.
10. Repeat step 5.9 until apoptotic cells are stained brown. Soak glass slides in water to stop the peroxidase reaction.
11. Enclose cells with water for observation.

6. Measure the Level of Phagocytosis of Apoptotic Cells

1. 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.
2. 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.

Wyniki

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 "Croquemort"^17,22 using a specific antibody^19,21, and apoptotic cells were stained by TUNEL in dispersed...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
whole swine serum	MP Biomedicals	55993	For bloking
Treff micro test tube(easy fit)  Dnase, Rnase free tube, 1.5 mL	TreffLab	96. 4625. 9. 01	For  homogenization
pellet mixer  1.5 mL	TreffLab	96. 7339. 9. 03	For  homogenization
Collagenase	Sigma-Aldrich	C-0130	For preparation of embryonic cells
Trypsin	Thermo Fisher SCIENTIFIC	27250-018	For preparation of embryonic cells
Kpl Anti-Rat IgG (H+L) Ab MSA, AP	KPL	475-1612	secondary antibody for stainig hemocytes with an anti-Croquemort antibody
5-bromo-4-chloro- 3-indolyl-phosphate	Roche	11383221001	BCIP, For staining of hemocytes
nitro blue tetrazolium	Roche	11383213001	NBT, For staining of hemocytes
Anti-Croquemort antibody			described previously in Manaka et al., J. Biol. Chem., 279, 48466-48476
Anti-GFP from mouse IgG1κ (clones 7.1 and 13.1)	Roche	11814460001	For staining of hemocytes
Goat Anti-Mouse IgG-AP Conjugate	Bio-Rad	170-6520	secondary antibody for stainig hemocytes with an anti-Croquemort antibody
Apop Tag Peroxidase In Situ Apoptosis Detection Kit	Millipore	S7100	For staining of apoptoitc cells. This kit includes Equilibration buffer, Reaction buffer, STOP/Wash buffer, TdT enzyme, and Anti-Digoxigenin-Peroxidase.
3,3'-diaminobenzidine tetrahydrichloride	nacalai tesque	11009-41	DAB, For staining of apoptoitc cells
Table of Fly Strains
Name	Stock center	Stock ID	Comments
w1118			Control flies, described in Freeman et al., Neuron, 38, 567-580
drprΔ5			drpr mutant, described in Freeman et al., Neuron, 38, 567-580
Itgbn2			Itgbn mutant, described in Devenport et al., Development, 131, 5405-5415
srpHemoGAL4 UAS-EGFP			described in Brückner et al., Dev. Cell., 7, 73-84
UAS-drpr-IR	VDRC	4833	-
UAS-Itgbn-IR	NIG-fly	1762R-1	-