The immunofluorescence staining technique is a golden standard biochemical method to detect. Flow cytometry flow cytometry is the technical process that allows for the individual measurements of cell fluorescence and light scattering. The flow cytometry protocols below provide detailed procedures for the treatment and staining of cells prior to using a flow cytometer. The method uses ethanol, a relatively mild solvent, to. A concentrationdependent selfassociation of nile red was also reported when the dye was added in excess, and aggregated dye precipitates were difficult to gate based on red channel fluorescence as the chlorophyll of microalgae using flow cytometry. The use of fluorescent nile red and bodipy for lipid measurement in microalgae. The peak emission fluorescence shifted from red to green with increasing hydrophobicity of the solvent and target staining lipid greenspan et al. Flow cytometric single cell analysis reveals heterogeneity. All staining procedures must be performed without direct exposure to intense light. Rapid detection of neutral lipid in green microalgae by. A flow cytometry method, to monitor peripheral lymphocytes phospholipidosis, has been set up using a single staining with nile red and double staining with nile red and anticd3 monoclonal antibody.
Various methods ranging from gravimetry to spectrophotometry are routinely used for qualitative analysis of extracted pha. A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument. Viability, as determined by lack of pi uptake, was greater than 95% in all nile red populations fig. Flow cytometry protocols flow cytometry guide biorad. The sample is focused to ideally flow one cell at a time through a laser beam and the light. Identification and quantification of microplastics using nile. May 30, 2014 initial techniques involving nile red were mostly qualitative or semiquantitative, combining the stain with singlecuvette spectrophotometry or flow cytometry. In most polar solvents, nile red will not fluoresce. Study of wood pitch emulsions interactions with nile red. Nile red is intensely fluorescent in a lipidrich environment while it has minimal fluorescence in aqueous media. Nile red stains intracellular lipid droplets yellow. I am doing nile red staining on glioma cells, harvesting around 80000cells with 0,5% trypsin edta, inactivation with fbs, transfer to facs tubes spinning 350g for 5mns, washing with 2ml pbs, spinning 350g for 5mns, adding 500ul nile red working solution for 15mn spinning 350g for 5mns, washing in pbs 1x. Applications of flow cytometry to clinical microbiology.
Combine the recommended quantity of each primary antibody in an appropriate volume of flow cytometry staining buffer so that the final staining volume is 100 l i. Pdf the use of fluorescent nile red and bodipy for lipid. A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using nile red nr, followed by densitybased extraction and filtration. Following is our recommended protocol for live cells. This information can be used to individually sort or separate subpopulations of cells. Pdf rapid flow cytometry nile red assessment of pha. Flow cytometry is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. As a result, researchers can use these particles for routine alignment, daytoday performance verification, and long term performance tracking of several flow cytometer channels in one run. Improved permeation protocols for staining recalcitrant microalgae. Overview ab228553 nile red staining kit is designed for the quantitative, fluorometric detection and measurement of intracellular lipid droplets using fluorescence microscopy, flow cytometry or a fluorescence microplate reader. In the assay, nile red dye is added to the cell samples and the dye selectively stains lipid droplets within the cells.
The staining protocol described in section 3 is designed for quantitative analysis, but it can. A simple and ef cient method for poly3hydroxybutyrate. There is a great need for accurate quantification of intracellular pha during bioprocess. Nile red is used to localize and quantitate lipids, particularly neutral lipid droplets within cells. Flow cytometry protocol flow cell signaling technology. Lipid droplets fluorescence assay kit cayman chemical. Please see the productspecific flow protocol on the product webpage for appropriate fixation and permeabilization conditions, and recommended. Nile red and 2nbdg are incompatible for the simultaneous. However, staining protocols with these dyes, especially with microal gae, vary. A flow cytometer, fluorescence microscope, or plate reader with the capacity to measure.
How to use nile red, a selective fluorescent stain for. A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using nile red nr, followed by. Mar 16, 2017 a new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using nile red nr, followed by densitybased extraction and filtration. A simple and rapid protocol for measuring neutral lipids in. Preparation of cells for flow cytometry for further information. Below, we briefly describe some of the applications of fcm in the field of microbiology, focusing on present or future applications in clinical microbiology. Nile red also known as nile blue oxazone is a lipophilic stain. A simple and rapid protocol for measuring neutral lipids. Image of nile red staining showing lipid droplets red. Adjust the cell suspension to a concentration of 1 x 10 7 cellsml with cold 4 o c pbsbsa buffer.
Run nile red with a fluorescence microscope or a flow cytometer. Flow cytometry facs protocols psr the bd facscalibur platform allows users to perform both cell analysis and cell sorting in a single benchtop system. In this paper, nile red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple. Discover more with our introduction to flow cytometry.
Principles and applications of flow cytometry table of contents. Recent improvements to the nile red staining method have been reported that bypass the initial shortcomings of the protocol10,11. See protocol ep7 fixation and immunofluorescence if. Nile red dimers and aggregates interfere with fluorescence intensity of neutral lipids. The following protocol can be used for staining cells to be assayed by flow cytometry best for suspension cells, microscopy, or plate reader detection. Quantification of intracellular polyhydroxyalkanoates by. Novel flow cytometric approach for the detection of adipocyte sub. Pdf improved nile red staining of nannochloropsis sp. Several authors report failure of efficient cell staining with nile red, caused by. Hcs lipidtox neutral lipid stains thermo fisher scientific. Nile red staining as a subsidiary method for microplastic.
It is an excellent vital stain for the detection of intracellular lipid droplets with fluorescence microscopy and flow cytometry. In addition, some classes of algae such as green algae have thick cell wells that are mostly impermeable to the dye, which limited the range of the technique 10. The following protocol can be used for staining cells to be assayed by. Flow cytometry is now a widely used method for analyzing the expression of cell surface and intracellular. Mar 12, 2015 a concentrationdependent selfassociation of nile red was also reported when the dye was added in excess, and aggregated dye precipitates were difficult to gate based on red channel fluorescence as the chlorophyll of microalgae using flow cytometry. The other protocols use fresh cells, so dyeing can be done right after sampling and washing. Yellow red fluorescence has been used mainly for lipid staining in various biological samples diaz et al. The effect of the ethanol, the solvent used for nile red, on nile red and the model pitch emulsion was also investigated with flow cytometry. The sample can be analyzed after exactly 30 min incubation time at room temperature in the dark. The use of fluorescent nile red and bodipy for lipid measurement. Blood has been collected from rats treated with amiodarone phospholipidogenic antiarrhythmic drug. Parameter ranges are given for protocol optimization. The fixed cells should be analyzed as soon as possible. Optimization of nile red is needed for different microalgae strains.
Flow cytometry was used to measure the phb content in cupriavidus necator formerly ralstonia eutropha 7 by measuring the change in the cellular light scattering properties caused by the formation of phb granules 8. In support of the microscopy and spectrofluorometry data shown here, we also performed a preliminary flow cytometry experiment with 2nbdg and nile red singly stained and costained adipocytes as flow cytometry can use compensation to reduce spillover from nile red when detecting 2nbdg. Pdf highthroughput screening of microalgae for use as a potential feedstock for biodiesel requires a reliable method for the rapid detection of. A more sensitive method consists of staining the cellular phb with nile red followed by analysis with either. Nile red staining as a sentinel screen introduction for. If analysis must be delayed, then the stained cells can be fixed with buffered paraformaldehyde for example, bd cytofix buffer, see the product tds for a detailed protocol for 30 minutes at 4c, washed, resuspended in stain buffer, and then stored at 4c protected from light.
Rapid flow cytometry nile red assessment of pha cellular content and heterogeneity in cultures of pseudomonas aeruginosa 47t2 ncib 40044 grown in waste frying oil. Hence, the present study aims to improvise the existing nile red based flow cytometry protocol. L nile red solution 1 mg nile red per ml acetone was added. Similar data were obtained with both lipidtox red and deep red neutral lipid stains. This process is performed at rates of thousands of cells per second. Better selectivity for cytoplasmic lipid droplets can be. A simple and rapid protocol for measuring neutral lipids in algal. In this paper, nile red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of auxenochlorella protothecoides, a green alga. Adherent cells prepare the nile red staining solution as directed and equilibrate to. Therefore, incubations need to be done in the dark. Proceed to step 8 for purified or biotinylated primary antibodies. The accumulation of cytoplasmic polyhydroxyalkanoates phas and the heterogeneity of bacterial populations were analysed by flow cytometry and syto and nile red staining in rhamnolipidproducing pseudomonas aeruginosa cultures grown in waste.
Rapid detection of neutral lipid in green microalgae by flow cytometry in combination with nile red stainingan improved technique. Monitoring the functionality and stress response of yeast. The use of fluorescent nile red and bodipy for lipid. Presently, more than 40,000 journal articles referencing flow cytometry have been published. This is one of the simplest and most common staining methods, where live or fixed cells are incubated with directly labeled antibodies against cell surface antigens.
Nile red is almost nonfluorescent in water and other polar solvents but undergoes fluorescence enhancement and large absorption and emission blue shifts in nonpolar environments excitationemission maxima 552636 nm in methanol. In this study, green emission fluorescence was sufficiently clear to enable. Introduction to flow cytometry flow cytometry is a popular laserbased technology. The system supports a wide variety of research and clinical applications and is complemented by a broad suite of intuitive software solutions to. Please refer to the applications section on the front page of product datasheet or product webpage to determine if this product is validated and approved for use in flow cytometry f. Rapid flow cytometry nile red assessment of pha cellular. Flow cytometry, with double staining with nile red and anti. These particles are very stable, since the fluorochromes are encapsulated inside. May 30, 2012 to address this, microalgae were stained with either the lipophilic dyes often used for lipid quantification nile red and bodipy or a lipophilic dye commonly used to stain neuronal cell membranes dio, and fluorescence was measured using flow cytometry. Whole blood samples may be used undiluted unless the cell count is high, e.
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