Phagocytosis Fluorescent Beads
Fluorescent monodisperse latex beads and a computer-centered spectrofluorimeter were used to devise a sensitive new assay for phagocytosis. These microspheres are packaged as 1 wv suspension in deionized DI water 1mL contains 10mg of fluorescent particles.
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This uptake can be tracked by using particles labeled with different fluorescent dyes such as hexidium iodide and Alexa Fluor 488 or genetically engineered bacteria expressing enhanced green fluorescent protein 46.. Bead dyes are spectrally matched to common lasers and are highly stable. Phagocytic particle uptake was calculated from data obtained by microscopy and flow cytometry utilizing commercially fluorescent beads of uniform 2 micrometer diameter size. Thus was not indicative of phagocytosis.
Phagocytosis has been widely studied by monitoring the uptake of fluorescent beads or bacteria of 1030 lm in diam-eter or length. Bangs fluorescent particles have been used in phagocytosis and neural retrograde transport studies and as markers for cell bound antigens. Flow cytometric evaluation of phagocytosis of fluorescent latex beads represents a simple rapid nonradioactive index of thyroid function in vitro.
Incubation time incubation temperature and beadcell ratio. Fluorescent beads are the most usual method-either by flow cytometry or microscopy. Beads bacterial and yeast particles labeled with pHrodo red and green were tested for their uptake by THP-1 cells and primary human macrophages by flow cytometry and high-content imaging.
To induce phagocytosis IgG-coated polystyrene beads with a diameter of 6067 µm were incubated with Laurdan-labelled macrophages. In this study we describe a. The engulfed fluorescent beads can be detected at the single-cell level.
Simplified description of the method and its applications Fluorescent latex beads are easily utilized to quantitate phagocytosis in cells with high endocytic rates such as LM fibroblasts. Learn More about Flow Cytometry Reference StandardsSection 232. We comprehensively evaluated and optimized phagocytosis assays using particles labeled with fluorescent pH-sensitive pHrodo dyes facilitating the specific detection of phagocytosed particles.
8772249 PubMed - indexed for MEDLINE Publication Types. Caymans Phagocytosis Assay Kit IgG FITC employs latex beads coated with fluorescently-labeled rabbit IgG as a probe for the measurement of the phagocytic process in vitro. If resources are limited or there is no access to flow cytometry simple microscopy of uptake of latex.
Phagocytosis was measured by direct counts of the percent phagocytic cells and the number of intracellular beads per 100 cells as seen by fluorescence microscopy. Another method used injection of fluorescent latex beads into rats followed by removal Address for correspondence. In addition the flow cytometric readout provides the.
GP images were pseudo-colored so that yellow to red indicates highly ordered membranes while green represents fluid membranes Figure 1A. A simple high-throughput flow cytometric assay was developed that uses THP-1 cells and fluorescent beads covalently-coupled with the malarial antigen VAR2CSA. The ability of FRTL-5 rat thyroid follicular cells to engulf latex beads by phagocytosis was evaluated using flow cytometry and compared to iodide trapping in response to selected growth factors s.
Here beads are selected with the FITC channel. PMNs collected from a Ficoll-Hypaque density gradient were incubated with the fluorescent beads in human plasma and gently washed in buffer to remove most of the extracellular beads. Phagocytosis is a fundamental process by which cells engulf extra-cellular particles with a size of 05 μm 1.
B Inside this population beads are highly fluorescent in all channels. Note that beads are located at the top of the graph. These fluorescent microspheres provide flow cytometry users the means to calibrate laser alignment compensation flow rate dynamic range and other daily adjustments.
Sandra Casinghino Pfizer Worldwide Research and Development Drug Safety Research and Development Groton CT 06340 USA. It has also been used to assess the uptake of latex beads by antigen presenting cells from porcine respiratory system. Specific endocytosis may simply be determined by measurement of the difference in fluorescent bead.
Detection of fluorescent beads in the flow cytometer for cell counting. Carboxylate-modified polystyrene latex beads fluorescent orange have been used to block phagocytosis by hemocytes in Drosophila. Phagocytic activity was stimulated by both cAMP-dependent and cAMP-independent pathways.
However to date no published method quanti-fies particle uptake in real time which is necessary to fully assess the kinetics of phagocytosis. A Side scatter area SSC-A versus forward scatter area FSC-A gating to obtain bead profiles. LM fibroblasts a transformed cell line with a high endocytic rate were exposed to fluoresbrite beads and the following parameters were investigated.
This uptake can be tracked by using particles labeled with different fluorescent dyes such as hexidium iodide and Alexa Fluor 488 or genetically engineered bacteria expressing enhanced green fluorescent protein 46.
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