Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

Summary

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

Abstract

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 × 10⁶ live hemocytes can be obtained from 200 Drosophila 3^rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3^rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Introduction

Drosophila melanogaster is a well-established model organism for the study of innate immunity¹. During the innate immune response, hemocytes play an important role in the response to pathogen challenge. Hemocytes are critical for encapsulating parasites, as well as having an important function in combating the pathogen through phagocytic action during fungal, viral, and bacterial infection^2,3.

In order to best understand the host's innate immune response to pathogenic microbial infection, it is important to visualize how the pathogen invades....

Protocol

1. Ex vivo infection

1. Medium and equipment
   1. Under sterile conditions, prepare fresh Drosophila Hemocyte Isolating Medium (DHIM) containing 75% Schneider's Drosophila medium with 25% Fetal Bovine Serum (FBS) and filter sterilize it.
   2. Layer 2-3 pieces of 10 cm x 10 cm paraffin film under a stereomicroscope.
   3. Prepare the glass capillary. Set the capillary puller heater to 55% of maximum. Pull the capillary tube to a sharp point of approximately 10 µm.
   4. Backfill the capillary with mineral oil.
   5. Assemble filled capillary tube onto the nanoinjector (Figure 1

Results

To collect live hemocytes for ex vivo infection, up to 3×10⁶ hemocytes were extracted from 200 Drosophila 3^rd instar larvae. To develop our method, a number of different techniques were attempted. Individual larval dissection would take up to 1.5 h, and an average of ~8000 cells were obtained using this method¹⁸, most of which were not alive by the end of collection. Next, we tried to extract hemolymph, which contained the he.......

Discussion

To better understand how host cells become infected, it is important to clarify the localization of pathogen in the cells, especially when experimenting on previously untested pathogen and cell type combinations⁴. While studying the cellular response cascade following infection can indicate productive pathogen invasion, the combination of imaging and cellular response data is essential to demonstrate pathogen invasion and infection. While reports showing 2D images of pathogen invasion into the hos.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Schneider's Drosophila Medium	Thermo Fisher Scientific (Gibco)	21720024	1.1.1), 2.1.2)
Fetal Bovine Serum	GE Healthcare Life Sciences  (HyClone)	SH30070.03HI	1.1.1), 2.1.2)
Filter (0.22 µL)	RESTEK	26158	1.1.1)
Strainer (100 µm)	Greiner bio-one	542000	1.2.1), 2)
Stereo microscope	Amscope	SM-1BSZ-L6W	1.2), 2)
Glass capillary	Fisher Scientific	21-171-4	1.1), 1.2), 2)
Capillary puller	Narishige International USA, Inc.	PC-10	1.1.3)
Mineral oil	Snow River Products		1.1.4)
Nanoinjector	Drummond Scientific Company	3-000-204	1.1), 1.2), 2.2)
Forceps	VWR	82027-402	1.1.5), 1.2), 2), 3.1.7)
CO2 delivery apparatus	Genesee Scientific	59-122BC	1.2), 2)
Trypan Blue	Thermo Fisher Scientific (Gibco)	15250061	1.3)
Hemocytometer	Hausser Scientific	3100	1.3)
24 well plate	Greiner bio-one	662160	1.4), 2.2)
Coxiella burnetii - mCherry	Dr. Heinzen, R.		1.4), 2.2)
Drosophila fruit juice plates	Cold Spring Harbor Protocols		2.1) http://cshprotocols.cshlp.org/content/2007/9/pdb.rec11113.full
Agar	Fisher Bioreagents	BP1423-500	2.1.1.1)
Methyl paraben	Amresco	0572-500G	2.1.1.2)
Absolute ethanol	Fisher Bioreagents	BP2818-500	2.1.1.2)
Welch's 100% Grape juice frozen concentrate, 340 mL	Amazon	B0025UJVGM	2.1.1.3)
Petri dishes, 10 x 35 mm	Fisher Scientific	08-757-100A	2.1.1.4)
Microscope cover glass	Fisher Scientific	12-545-80	1.4.4), 2.2.2)
Yeast, Bakers Dried Active	MP Biomedicals	0210140001	2.1) Add 2 parts of water to 1 part of yeast (v/v)
Tungsten needle	Fine Science Tools	10130-20	2.1)
Holding forceps	VWR	HS8313	2.1)
Paraformaldehyde	Fisher Scientific	FLO4042-500	3.1.3)
Triton X-100	Fisher Scientific	BP151-500	3.1.3)
Bovine Serum Albumin	Fisher Scientific	BP9706-100	3.1.3)
4',6-diamidino-2-phenylindole	Thermo Fisher Scientific	62247	3.1.4)
Antifade mounting medium	Thermo Fisher Scientific	P36930	3.1.6)
Confocal microsope	Leica	TCS SP8-X White Light Confocal Laser Scanning Microscope	3.2)
3D imaging reconstruction software	Leica	LASX with 3D visualization module	3.3)
Microscope slides	Fisher Scientific	12-552-3	3.1.6)
Invertebrate iridescent virus 6 (IIV6)	Dr. Teixeria, L.		4) PLoS Biol, 6 (12), 2753-2763, doi: 10.1371/journal.pbio.1000002, (2008)
Listeria monocytogenes	ATCC	strain: 10403S	4) Listeria monocytogenes strain 10403S (Bishop and Hinrichs, 1987) was grown in Difco Brain-heart infusion (BHI) broth (BD Biosciences) containing 50 µg/ml streptomycin at 30 °C.
DNase I	Thermo Fisher Scientific(Invitrogen)	18068015	gDNA degradation
cDNA Synthesis Kit	Bio-Rad	1708891	cDNA synthesis
IIV6_193R_F	IDT		qRT-PCR, 5'- TCT TGT TTT CAG AAC CCC ATT -3'
IIV6_193R_R	IDT		qRT-PCR, 5'- CAC GAA GAA TGA CCA CAA GG -3'
RpII_qRTPCR_fwd	SIGMA-ALDRICH		qRT-PCR, 5'- GAA GCG TTT CTC CAA ACG -AG
RpII_qRTPCR_rev	SIGMA-ALDRICH		qRT-PCR, 5'- TTG AGC GTA AGC ATC ACC -TG
SYBR Green qRT-PCR reagent	Thermo Fisher Scientific	K0251, K0252, K0253	qRT-PCR
Real-Time PCR System	Thermo Fisher Scientific	4351107, 7500 Software v2.0	qRT-PCR
Anti-Listeria monocytogenes antibody	abcam	ab35132	Western blot
Anti-Actin antibody produced in rabbit	SIGMA-ALDRICH	A2066	Western blot
Anti-Rabbit IgG (H+L), HRP Conjugate	Promega	W4011	Western blot