Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 c....

Results

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 embryon.......

Discussion

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.......

Materials

List of materials used in this article

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	-