An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Abstract

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.

Heat-killed Mycobacterium tuberculosis prime-boost vaccination induces myeloid-derived suppressor cells with spleen dendritic cell–killing capability

“…Software-based quantification of TUNEL + /CD11c + cells. Images were processed with the StrataQuest software (TissueGnostics) as described previously (32). Using that software, 3 markers were applied: CD11c, TUNEL, and B220.…”

“…In contrast, after 24 hours TUNEL + cells were readily detectable in the T cell areas of spleens in CFA/CFA plus LPS/IFN-γinjected mice ( Figure 5E). To quantify DC apoptosis in the T cell areas, we used StrataQuest software with the same markers and costained T cells obtained from splenic sections of WT and Nos2 -/mice by TUNEL (32). Indeed, a substantial proportion of spleen sections showed increased frequencies of CD11c + TUNEL + cells in the T cell areas at 24 hours.…”

Immune Cell and Cell Cluster Phenotyping, Quantitation, and Visualization Using In Silico Multiplexed Images and Tissue Cytometry

“…A line border was created to encase the filled mask to demark the border of the nucleus. A cellular mask algorithm was used to identify the biomarker for the cell phenotype . A ring mask was used with a −0.32 μm interior radius, 0.63 μm exterior radius, 1.26 μm maximum growing step, and a five grey level background threshold.…”

PI3K: A master regulator of brain metastasis‐promoting macrophages/microglia

“…The analysis was performed with TissueQuest Analysis software (TissueGnostics), as described before (Schmid et al, 2015). The analysis was performed with TissueQuest Analysis software (TissueGnostics), as described before (Schmid et al, 2015).…”

“…Cells were fixed in ice-cold acetone and incubated with Phalloidin-TRITC (Sigma), followed by staining with GS-IB4 Alexa 647 (Life Technologies, Carlsbad, USA), counterstained with DAPI (Sigma), and scanned with the TissueFAXS i-plus imaging system (TissueGnostics, Vienna, Austria). The analysis was performed with TissueQuest Analysis software (TissueGnostics), as described before (Schmid et al, 2015). For detailed instrumental settings please refer to Supporting Information.…”