An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry.
Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Maximilian
M
Schmid
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Bianca
B
Dufner
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Julius
J
Dürk
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Konstanze
K
Bedal
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Kristina
K
Stricker
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Lukas Ali
LA
Prokoph
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Christoph
C
Koch
Department of Gynecology and Obstetrics, University of Regensburg, Regensburg, Germany.
Anja K
AK
Wege
Division of Immunology, Paul-Ehrlich-Institute, Langen, Germany.
Henner
H
Zirpel
Department of Gynecology and Obstetrics, University of Regensburg, Regensburg, Germany; Institute of Immunology, University Medical Center of the Johannes Gutenberg University of Mainz, Mainz, Germany.
Ger
G
van Zandbergen
TissueGnostics GmbH, Vienna, Austria.
Rupert
R
Ecker
TissueGnostics GmbH, Vienna, Austria.
Bogdan
B
Boghiu
Institute of Immunology, University of Regensburg, Regensburg, Germany.
Uwe
U
Ritter
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D019295:Computational Biology / Q000379:methods*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D006790:Host-Parasite Interactions; D006801:Humans; D019044:Image Cytometry / Q000379:methods*; D007211:Indoles / Q000737:chemistry; D042541:Intracellular Space / Q000469:parasitology; D018320:Leishmania major / Q000235:genetics / Q000502:physiology*; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology; D008198:Lymph Nodes / Q000469:parasitology; D008264:Macrophages / Q000378:metabolism / Q000469:parasitology*; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012984:Software
D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI