A new method for yeast phagocytosis analysis by flow cytometry (original) (raw)
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Flow cytometry distinction between adherent and phagocytized yeast particles
Cytometry, 1994
Our laboratory recently developed a light microscopy staining technique that provides a mean to distinguish between yeast that are simply bound to the surface of macrophages and yeast that have actually been phagocytized by macrophages (7). We adapted this technique by using fluorescent probes in order to test phagocytic activity by flow cytometry. Thus we are able to distinguish unambiguously extracellular from intracellular yeast during phagocytosis with the fast rate of flow cytometry (-200 cells/s). The fluorescence quenching induced by a 1% tannic acid solution (wlv) can be applied to any FITC-labeled, heat-killed yeast cell or bacteria. The yeast cells already engulfed in the macrophage remain with their native fluorescence (internal and external pH equilibrated by 50 pM monensin 30 minI4"C) protected from the action of tannic acid, a nonmembrane permeable molecule. The results presented here validate this new technique. An application is presented showing the inhibition of endocytosis by cytochalasin-B.
Use of SYTOX green dye in the flow cytometric analysis of bacterial phagocytosis
Cytometry, 2002
BackgroundFluorescein isothiocyanate (FITC) is used widely to label the targets used in flow cytometric phagocytosis assays. Unfortunately, the fluorescence intensity of phagocytosed FITC-labeled targets is influenced by changes in intracellular pH level, making quantitative measurements with this fluorophore problematic. We describe the use of SYTOX green nucleic acid stain to measure phagocytosis by flow cytometry.Fluorescein isothiocyanate (FITC) is used widely to label the targets used in flow cytometric phagocytosis assays. Unfortunately, the fluorescence intensity of phagocytosed FITC-labeled targets is influenced by changes in intracellular pH level, making quantitative measurements with this fluorophore problematic. We describe the use of SYTOX green nucleic acid stain to measure phagocytosis by flow cytometry.MethodsSuspensions of isopropyl alcohol-permeabilized Escherichia coli DH5α were stained with the SYTOX green dye and then incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytosis was determined by gating the cells.Suspensions of isopropyl alcohol-permeabilized Escherichia coli DH5α were stained with the SYTOX green dye and then incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytosis was determined by gating the cells.ResultsResults are expressed as percentage of phagocyte-associated green fluorescent cells. The validity of the method was shown by the effects of a phagocytosis inhibitor (incubation at 4°C) or enhancer (gamma interferon [IFN- γ] treatment) being accurately assessed with this assay.Results are expressed as percentage of phagocyte-associated green fluorescent cells. The validity of the method was shown by the effects of a phagocytosis inhibitor (incubation at 4°C) or enhancer (gamma interferon [IFN- γ] treatment) being accurately assessed with this assay.ConclusionsThe method described was reproducible and provides an advantageous alternative to the use of FITC to label bacteria for the flow cytometric measurement of target uptake by phagocytic cells. Cytometry 48:93–96, 2002. © 2002 Wiley-Liss, Inc.The method described was reproducible and provides an advantageous alternative to the use of FITC to label bacteria for the flow cytometric measurement of target uptake by phagocytic cells. Cytometry 48:93–96, 2002. © 2002 Wiley-Liss, Inc.
Application of Flow Cytometry to Saccharomyces cerevisiae Population Analysis
CHIMIA, 2005
This study was focused on the development and the application of a rapid and reliable staining method for the characterisation of Saccharomyces cerevisiae cells. The experiments were carried out during the shaken flask batch cultivation of yeasts on YEPD medium under aerobic conditions at 27 °C. Stained samples were analysed with an epifluorescence microscope or by employing a flow cytometer. Three different fluorescent probes such as propidium iodide (PI), fluorescein diacetate (FDA), and fluorescein isothiocyanate (FITC) were used for staining. PI was used to determine cell viability in a native sample and DNA content in a sample fixed by ethanol. To assess protein distribution in the yeast population the FITC amine-reactive probe was used. Instantaneous cell enzyme activity was measured as the amount of fluorescein liberated from FDA by intracellular esterase activity.
Applied and Environmental Microbiology, 2009
The measurement of yeast's intracellular pH (ICP) is a proven method for determining yeast vitality. Vitality describes the condition or health of viable cells as opposed to viability, which defines living versus dead cells. In contrast to fluorescence photometric measurements, which show only average ICP values of a population, flow cytometry allows the presentation of an ICP distribution. By examining six repeated propagations with three separate growth phases (lag, exponential, and stationary), the ICP method previously established for photometry was transferred successfully to flow cytometry by using the pH-dependent fluorescent probe 5,6-carboxyfluorescein. The correlation between the two methods was good ( r 2 = 0.898, n = 18). With both methods it is possible to track the course of growth phases. Although photometry did not yield significant differences between exponentially and stationary phases ( P = 0.433), ICP via flow cytometry did ( P = 0.012). Yeast in an exponenti...
Rapid susceptibility testing of fungi by flow cytometry using vital staining
Journal of clinical microbiology
A 1-h assay for antifungal susceptibility testing measuring the impairment of fungal metabolic activity was developed. Yeast viability was analyzed by flow cytometry with a novel fluorescent probe, FUN-1, which emits a red fluorescence when the yeast is metabolically active. For nine Candida albicans strains tested, this method yielded results comparable to those obtained by the standard M27 procedure for amphotericin B, flucytosine, fluconazole, and ketoconazole. Whether the flow cytometry antifungal susceptibility test results correlate with the in vivo activities of the drugs remains to determined.
A novel flow cytometric protocol for assessment of yeast cell adhesion
Cytometry Part A, 2012
Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner. '
Biotechnology and Bioengineering, 2004
This article describes a new method for the early detection of alcoholic fermentation arrest. This methodology is based on the flow cytometric assessment of Saccharomyces cerevisiae yeasts stained with a carboxyfluorescein diacetate fluorescent viability probe. Multicomponent analysis of viable cell distribution constitutes a promising new tool to describe physiological and dynamic changes to heterogeneous viable populations during alcoholic fermentation, through its ability to discriminate between successful processes and those ending prematurely. This framework, which is based on the comparison of cytometric histogram descriptors' combinations that can be related to simple physiological significance comparison, quickly and simply, allows testing yeasts for their fermentation ability and can be used to detect any kind of viability loss so that fermentation arrest can be avoided. B 2004
A novel flow cytometric method for quantifying phagocytosis of apoptotic cells
Cytometry, 1997
Many eukaryotic cell types are capable of specific recognition and phagocytosis of apoptotic cells, and there is increasing interest in the mechanisms involved in this process. To facilitate analysis of these mechanisms, we designed a novel fluorescence-based method to quantify phagocytosis in vitro using endothelial cell engulfment of apoptotic cells as a model. The B-cell line WEHI-231 was labeled with the fluorophore 5-(&-6)-carboxytetramethylrhodamine-succinimidyl-ester (TAMRA) and then induced to undergo apoptosis by crosslinking cell surface immunoglobulin. An endothelial cell line was subsequently allowed to ingest these TAMRA-labeled apoptotic lymphocytes. After 24 h, non-bound lymphocytes were removed and the monolayers were dissociated. Any nonphagocytosed lymphocytes that remained tightly bound to the endothelial cells were then indirectly immunofluorescein labeled for the pan leukocyte-specific marker CD45. Flow cytometric analysis of the cells distinguished three endothelial cell populations: 1) endothelial cells with surface bound lymphocytes (TAMRA + CD45 + ); 2) endothelial cells containing phagocytosed apoptotic lymphocytes (TAMRA + CD45 − ); and 3) endothelial cells that were not associated with lymphocytes. The identification of these populations was verified by confocal microscopy of sorted cells. The method described herein will facilitate detailed studies on phagocytic recognition of apoptotic cells and should have broad applications to other phagocytic cell systems.