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

In this study, we determine whether the fluorescence labeling processes themselves influence the results of the phagocytosis measurements. Here, alveolar macrophages, which form one of the most important compartments of the innate immune system, were used as an example of host cells, whereas Aspergillus fumigatus and Lichtheimia corymbifera that cause aspergillosis and mucormycosis, respectively, were studied as examples for pathogens. At first, our study investigated the importance of the sequence of steps of the fixation process when preparing the confrontation assay sample for microscopy studies. Here we showed that applying the fixation agent before the counter-staining causes miscalculations during the determination of the phagocytic measures. Furthermore, we also found that staining the macrophages with various concentrations of DID, as a typical membrane label, in most cases altered the capability of macrophages to phagocytose FITC-stained A. fumigatus and L. corymbifera spores in comparison with unlabeled macrophages. This effect of the DID staining showed a differential character dependent upon the labeling status and the specific type of pathogen. Moreover, labeling the spores of A. fumigatus and L. corymbifera with FITC increased the phagocytic measures during confrontation with unlabeled macrophages when compared to label-free spores. Overall, our study confirms that the staining process itself may significantly manipulate the quantitative outcome of the confronta...
Source: Frontiers in Microbiology - Category: Microbiology Source Type: research