Journal article
Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis
Journal of visualized experiments : JoVE, Vol.2018(135)
05/24/2018
Handle:
https://hdl.handle.net/2376/100408
PMCID: PMC6101378
PMID: 29889203
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 × 10
live hemocytes can be obtained from 200 Drosophila 3
instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3
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.
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Details
- Title
- Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis
- Creators
- Aoi Hiroyasu - School of Molecular Biosciences, College of Veterinary Medicine, Washington State UniversityDavid C DeWitt - Integrative Physiology and Neuroscience, College of Veterinary Medicine, Washington State UniversityAlan G Goodman - School of Molecular Biosciences, College of Veterinary Medicine, Washington State University; agoodman@vetmed.wsu.edu
- Publication Details
- Journal of visualized experiments : JoVE, Vol.2018(135)
- Academic Unit
- Molecular Biosciences, School of
- Publisher
- United States
- Grant note
- R00 AI106963 / NIAID NIH HHS R21 AI128103 / NIAID NIH HHS
- Identifiers
- 99900546621101842
- Language
- English
- Resource Type
- Journal article