Name: constant pressure-controlled extrusion method for the preparation of nano-sized lipid vesicles
Uploaded: 2012-06-22T13:00:00
Description: Watch this Scientific Journal Video about Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles at JoVE.com

This work was supported by the Howard Hughes Medical Institute (HHMI) Collaborative Innovation Award. L. A. M. was supported by the Signaling and Cellular Regulation National Institute of Health training grant (T32 GM008759) and the NIH Ruth L. Kirschstein Pre-Doctoral Fellow (CA165349-01). We would like to thank Prof. Michael Stowell (CU Boulder), Prof. Douglas Rees and Prof. Rob Phillips (Caltech) for their invaluable comments.

1. Liposome Preparation
<ol>
	<li>Retrieve a 20 mL glass vial with a Teflon-lined cap.</li>
	<li>Clean all glassware and syringes with chloroform prior to use to prevent contamination.</li>
	<li>Transfer 100 &mu;L of reagent grade chloroform to the glass vial using a 250 &mu;L air-tight glass syringe.</li>
	<li>Add 30 &mu;L of reagent grade methanol to the same glass vial using a 100 &mu;L air-tight glass syringe.</li>
	<li>To prepare a 2 mM 7:1.5:1.5 phosphatidylcholine (POPC): phosphatidylethanolamine (POPE): cholest...

<ol>
	<li>Connor, J., Bucana, C., Fidler, I. J., Schroit, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+expression+of+phosphatidylserine+in+mammalian+plasma+membranes:+quantitative+assessment+of+outer-leaflet+lipid+by+prothrombinase+complex+formation.">Differentiation-dependent expression of phosphatidylserine in mammalian plasma membranes: quantitative assessment of outer-leaflet lipid by prothrombinase complex formation.</a> Proc. Natl. Acad. Sci. U.S.A. 86, 3184-3188 (1989).</li><li>Smith, S. A., Morrissey, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+incorporation+of+tissue+factor+into+liposomes.">Rapid and efficient incorporation of tissue factor into liposomes.</a> J. Thromb. Haemost. 2, 1155-1162 (2004).</li><li>Hui, E., Johnson, C. P., Yao, J., Dunning, F. M., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin-mediated+bending+of+the+target+membrane+is+a+critical+step+in+Ca(2+)-regulated+fusion.">Synaptotagmin-mediated bending of the target membrane is a critical step in Ca(2+)-regulated fusion.</a> Cell. 138, 709-721 (2009).</li><li>Loughrey, H. C., Choi, L. S., Cullis, P. R., Bally, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+procedures+for+the+coupling+of+proteins+to+liposomes.">Optimized procedures for the coupling of proteins to liposomes.</a> J. Immunol. Methods. 132, 25-35 (1990).</li><li>Mui, B., Chow, L., Hope, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrusion+technique+to+generate+liposomes+of+defined+size.">Extrusion technique to generate liposomes of defined size.</a> Methods Enzymol. 367, 3-14 (2003).</li><li>Gruner, S. M., Cullis, P. R., Hope, M. J., Tilcock, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+polymorphism:+the+molecular+basis+of+nonbilayer+phases.">Lipid polymorphism: the molecular basis of nonbilayer phases.</a> Annu. Rev. Biophys. Biophys. Chem. 14, 211-238 (1985).</li><li>Guven, A., Ortiz, M., Constanti, M., O'Sullivan, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+efficient+method+for+the+size+separation+of+homogeneous+fluorescein-encapsulating+liposomes.">Rapid and efficient method for the size separation of homogeneous fluorescein-encapsulating liposomes.</a> J. Liposome. Res. 19, 148-154 (2009).</li><li>Zimmerberg, J., Kozlov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+proteins+produce+cellular+membrane+curvature.">How proteins produce cellular membrane curvature.</a> Nature Reviews Molecular Cell Biology. 7, 9-19 (2006).</li><li>Chonn, A., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+liposomal+drug-delivery+systems.">Recent advances in liposomal drug-delivery systems.</a> Curr. Opin. Biotechnol. 6, 698-708 (1995).</li><li>Mayer, L. D., Hope, M. J., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicles+of+variable+sizes+produced+by+a+rapid+extrusion+procedure.">Vesicles of variable sizes produced by a rapid extrusion procedure.</a> Biochim. Biophys. 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Using the Avestin Liposofast LF-50 Extruder, we demonstrated how small-sized, synthetic liposomes are prepared through a pressure-controlled system. It is important to note that multilamellar vesicles form spontaneously following liposome hydration, which may lead to production of smaller nanoparticles. These small multilamellar vesicles will inevitably flow through the larger polycarbonate membrane pore size, causing heterogeneity in solutions of unilamellar vesicles produced by a large filter pore. Therefore, it is rec...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Sigma-Aldrich</td>
 <td>02432-25ML</td>
 <td>95% stabilizers</td>
 </tr>
 <tr>
 <td>High Grade Methanol</td>
 <td>Sigma-Aldrich</td>
 <td>179337-4L</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Liposofast LF-50 Extruder</td>
 <td>Avestin, Inc.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phospholipids</td>
 <td>Avanti Polar Lipids</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polycarbonate Pores</td>
 <td>Avestin, Inc.</td>
 <td>&nbsp;</td>
 <td>25 mm diameter</td>
 </tr>
 <tr>
 <td>Drain discs PE</td>
 <td>Avestin, Inc.</td>
 <td>230600</td>
 <td>25 mm diameter</td>
 </tr>
 </tbody>
</table>

constant pressure-controlled extrusion method for the preparation of nano-sized lipid vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

Watch this Scientific Journal Video about Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles at JoVE.com

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

constant-pressure-controlled-extrusion-method-for-preparation-nano

Research

JoVE Journal

Bioengineering

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...