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 x 10(6) live
hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo
infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd
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.