Quantitative Impact of Cell Membrane Fluorescence Labeling on Phagocytosis Measurements in Confrontation Assays

• DOI: 10.3389/fmicb.2020.01193
• Publication Date: 2020-06-05T04:26:17Z
• Authors: Zoltan Cseresnyes; Mohamed I. Abdelw...; Hans-Martin Dahse; Kerstin Voigt; Marc Thilo Figge
• Keywords: Diagnosis and Management of Fungal Infections; 0301 basic medicine; Cell biology; automated image analysis; Immunology; FOS: Health sciences; Microbiology; Biochemistry; 03 medical and health sciences; Bacterial Biofilms and Quorum Sensing Mechanisms; Role of Neutrophil Extracellular Traps in Immunity; Phagocytosis; Biochemistry, Genetics and Molecular Biology; Health Sciences; Genetics; confrontation assay; Flow cytometry; Molecular Biology; Biology; spinning disk confocal microscopy; Immunology and Microbiology; Staining; FOS: Clinical medicine; Aspergillus fumigatus; Innate immune system; Degranulation; phagocytosis; Life Sciences; Quorum Sensing; Spore; QR1-502; Infectious Diseases; Immune system; FOS: Biological sciences; Medicine; host–pathogen interaction; label-free imaging; Receptor

Phagocytosis is series of steps where the pathogens and immune cells interact during an invasion. This starts with adhesion process between host pathogen cells, followed by engulfment pathogens. Many analytical methods that are applied to characterize phagocytosis based on imaging host-pathogen confrontation assays rely fluorescence labeling cells. However, potential effect membrane quantitative results has not been studied in detail. In this study, we determine whether processes themselves influence measurements. Here, alveolar macrophages, which form one most important compartments innate system, were used as example whereas Aspergillus fumigatus Lichtheimia corymbifera cause aspergillosis mucormycosis, respectively, examples for At first, our study investigated importance sequence fixation when preparing assay sample microscopy studies. Here showed applying agent before counter-staining causes miscalculations determination phagocytic measures. Furthermore, also found staining macrophages various concentrations DID, a typical label, cases altered capability phagocytose FITC-stained A. L. spores comparison unlabeled macrophages. DID differential character dependent upon status specific type pathogen. Moreover, FITC increased measures compared label-free spores. Overall, confirms itself may significantly manipulate outcome assay. As result developed user-friendly image analysis tool analyses both without Our algorithm saves experimental work effort time, provides more precise calculating measures, delivers convenient biologists monitor interactions they happen artifacts imposes biological interactions.