Extracellular vesicles (EVs), including ‘microvesicles’ and ‘exosomes’, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.
24 Related JoVE Articles!
Quantification of Atherosclerotic Plaque Activity and Vascular Inflammation using [18-F] Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT)
Institutions: University of Pennsylvania, Perelman School of Medicine, University of Pennsylvania, Perelman School of Medicine, University of Pennsylvania, Perelman School of Medicine.
Conventional non-invasive imaging modalities of atherosclerosis such as coronary artery calcium (CAC)1
and carotid intimal medial thickness (C-IMT)2
provide information about the burden of disease. However, despite multiple validation studies of CAC3-5
, and C-IMT2,6
, these modalities do not accurately assess plaque characteristics7,8
, and the composition and inflammatory state of the plaque determine its stability and, therefore, the risk of clinical events9-13
F]-2-fluoro-2-deoxy-D-glucose (FDG) imaging using positron-emission tomography (PET)/computed tomography (CT) has been extensively studied in oncologic metabolism14,15
. Studies using animal models and immunohistochemistry in humans show that FDG-PET/CT is exquisitely sensitive for detecting macrophage activity16
, an important source of cellular inflammation in vessel walls. More recently, we17,18
and others have shown that FDG-PET/CT enables highly precise, novel measurements of inflammatory activity of activity of atherosclerotic plaques in large and medium-sized arteries9,16,19,20
. FDG-PET/CT studies have many advantages over other imaging modalities: 1) high contrast resolution; 2) quantification of plaque volume and metabolic activity allowing for multi-modal atherosclerotic plaque quantification; 3) dynamic, real-time, in vivo
imaging; 4) minimal operator dependence. Finally, vascular inflammation detected by FDG-PET/CT has been shown to predict cardiovascular (CV) events independent of traditional risk factors21,22
and is also highly associated with overall burden of atherosclerosis23
. Plaque activity by FDG-PET/CT is modulated by known beneficial CV interventions such as short term (12 week) statin therapy24
as well as longer term therapeutic lifestyle changes (16 months)25
The current methodology for quantification of FDG uptake in atherosclerotic plaque involves measurement of the standardized uptake value (SUV) of an artery of interest and of the venous blood pool in order to calculate a target to background ratio (TBR), which is calculated by dividing the arterial SUV by the venous blood pool SUV. This method has shown to represent a stable, reproducible phenotype over time, has a high sensitivity for detection of vascular inflammation, and also has high inter-and intra-reader reliability26
. Here we present our methodology for patient preparation, image acquisition, and quantification of atherosclerotic plaque activity and vascular inflammation using SUV, TBR, and a global parameter called the metabolic volumetric product (MVP). These approaches may be applied to assess vascular inflammation in various study samples of interest in a consistent fashion as we have shown in several prior publications.9,20,27,28
Medicine, Issue 63, FDG-PET/CT, atherosclerosis, vascular inflammation, quantitative radiology, imaging
Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles
Institutions: University of Colorado Boulder, University of Colorado Boulder.
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 <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
, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter <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 <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.
Bioengineering, Issue 64, Biomedical Engineering, Liposomes, particle extrusion, nano-sized vesicles, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA)
Particle Agglutination Method for Poliovirus Identification
Institutions: National Institute of Infectious Diseases, Fujirebio Inc..
In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).
Immunology, Issue 50, Poliovirus, identification, particle agglutination, virus receptor
Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels
Institutions: University Duesseldorf.
Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1
have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2
, and in 2004 Candiano et al
. established Blue Silver
using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3
. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al.
in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4
. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.
Basic Protocols, Issue 30, protein in-gel detection, Coomassie G-250, colloidal staining, 1- and 2-dimensional SDS-gels
Method for Measurement of Viral Fusion Kinetics at the Single Particle Level
Institutions: Harvard Medical School, Harvard Medical School.
Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro,
two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 μm2
area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.
Biomedical Engineering, Issue 31, Viral fusion, membrane fusion, supported lipid bilayer, biophysics, single molecule
Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro
method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo
experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e.
smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo
. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro
smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries
Institutions: University of Missouri, Dalton Cardiovascular Research Center.
The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.
endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+
signaling, which leads to production of diffusible autacoids (e.g.
nitric oxide and arachidonic acid metabolites)1-3
that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+
signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ
to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7
. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.
Basic Protocol, Issue 81, endothelial tubes, microcirculation, calcium signaling, resistance vasculature, Confocal microscopy
Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates
Institutions: Cytori Therapeutics Inc, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA, David Geffen School of Medicine at UCLA.
In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.
Cellular Biology, Issue 79, Adipose Tissue, Stem Cells, Humans, Cell Biology, biology (general), enzymatic digestion, collagenase, cell isolation, Stromal Vascular Fraction (SVF), Adipose-derived Stem Cells, ASCs, lipoaspirate, liposuction
Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography
Institutions: Loyola University Chicago.
Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+
), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g.
calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g.
to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.
Physiology, Issue 67, Molecular Biology, Medicine, Anatomy, Vascular smooth muscle, mesenteric artery, patch clamp, Kv channel, vasoconstriction, electrophysiology
Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice
Institutions: Northwestern University Feinberg School of Medicine.
Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26
that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 μM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.
Basic Protocol, Issue 80, Muscle, Smooth, Vascular, Cardiovascular Abnormalities, Hypertension, Pulmonary, vascular smooth muscle, pulmonary hypertension, development, phosphodiesterases, cGMP, immunostaining
Localized RNAi and Ectopic Gene Expression in the Medicinal Leech
Institutions: University of California San Diego - UCSD, University of California San Diego - UCSD.
In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to perturb gene expression patterns in selected segments of leech embryos, leaving the untreated segments as internal controls.
The pneumatic capillary gun can be used to reach internal layers of cells at early stages of development without opening the specimen. As a method for localized introduction of substances into living tissues, the biolistic delivery with the gun has several advantages: it is fast, contact-free and non-destructive. In addition, a single capillary gun can be used for independent delivery of different substances. The delivery region can have lateral dimensions of ~50-150 µm and extends over ~15 µm around the mean penetration depth, which is adjustable between 0 and 50 µm. This delivery has the advantage of being able to target a limited number of cells in a selected location intermediate between single cell knock down by microinjection and systemic knockdown through extracellular injections or by means of genetic approaches.
For knocking down or knocking in the expression of the axon guidance molecule Netrin, which is naturally expressed by some central neurons and in the ventral body wall, but not the dorsal domain, we deliver molecules of dsRNA or plasmid-DNA into the body wall and central ganglia. This procedure includes the following steps: (i) preparation of the experimental setup for a specific assay (adjusting the accelerating pressure), (ii) coating the particles with molecules of dsRNA or DNA, (iii) loading the coated particles into the gun, up to two reagents in one assay, (iv) preparing the animals for the particle delivery, (v) delivery of coated particles into the target tissue (body wall or ganglia), and (vi) processing the embryos (immunostaining, immunohistochemistry and neuronal labeling) to visualize the results, usually 2 to 3 days after the delivery.
When the particles were coated with netrin dsRNA, they caused clearly visible knock-down of netrin expression that only occurred in cells containing particles (usually, 1-2 particles per cell). Particles coated with a plasmid encoding EGFP induced fluorescence in neuronal cells when they stopped in their nuclei.
Neuroscience, Issue 14, leech, netrin, axon guidance, development, mechanosensory neurons, gene gun, RNAi
Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation
Institutions: University of Washington, Fred Hutchinson Cancer Research Center , University of Washington, Fred Hutchinson Cancer Research Center , Fred Hutchinson Cancer Research Center .
A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1
However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2
However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3
A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8
The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10
The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11
Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10
The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.
Bioengineering, Issue 55, Surface plasmon, optical trapping, optical tweezers, plasmonic trapping, cell manipulation, optical manipulation
Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins
Institutions: George Mason University, Ceres Nanosciences.
Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.
Bioengineering, Issue 90, biomarker, hydrogel, low abundance, mass spectrometry, nanoparticle, plasma, protein, urine
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125
I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125
I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
Graphene Coatings for Biomedical Implants
Institutions: Clemson University, East Carolina University, Clemson University, Clemson University.
Atomically smooth graphene as a surface coating has potential to improve implant properties. This demonstrates a method for coating nitinol alloys with nanometer thick layers of graphene for applications as a stent material. Graphene was grown on copper substrates via
chemical vapor deposition and then transferred onto nitinol substrates. In order to understand how the graphene coating could change biological response, cell viability of rat aortic endothelial cells and rat aortic smooth muscle cells was investigated. Moreover, the effect of graphene-coatings on cell adhesion and morphology was examined with fluorescent confocal microscopy. Cells were stained for actin and nuclei, and there were noticeable differences between pristine nitinol samples compared to graphene-coated samples. Total actin expression from rat aortic smooth muscle cells was found using western blot. Protein adsorption characteristics, an indicator for potential thrombogenicity, were determined for serum albumin and fibrinogen with gel electrophoresis. Moreover, the transfer of charge from fibrinogen to substrate was deduced using Raman spectroscopy. It was found that graphene coating on nitinol substrates met the functional requirements for a stent material and improved the biological response compared to uncoated nitinol. Thus, graphene-coated nitinol is a viable candidate for a stent material.
Biomedical Engineering, Issue 73, Bioengineering, Medicine, Biophysics, Materials Science, Physics, Pharmacology, Toxicology, Surgery, Chemistry and Materials (General), graphene, biomedical implants, surface modification, chemical vapor deposition, protein expression, confocal microscopy, implants, stents, clinical
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro
model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2
on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3
cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro
BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice
Institutions: Hunter College, CUNY.
Efficient expression of transgenes in vivo
is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).
Genetics, Issue 87, hydrodynamic gene delivery, hydrodynamics-based transfection, mouse, gene therapy, plasmid DNA, transient gene expression, tail vein injection
Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun
Institutions: University of Toronto, MRC-Laboratory of Molecular Biology, Cambridge, UK.
Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo
biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun's firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene's effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.
Neuroscience, Issue 92, Biolistics, gene gun, organotypic brain slices, Diolistic, gene delivery, staining
Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles
Institutions: The Pennsylvania State University.
In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma polymerization of isopropanol on various solid substrates, including silica and potassium chloride. This versatile technique is used to treat nanoparticles and nanopowders with sizes ranging from 37 nm to 1 micron, by depositing films whose thickness can be anywhere from 1 nm to upwards of 100 nm. Dissolution of the core allows us to study the rate of permeation through the film. In these experiments, we determine the diffusion coefficient of KCl through the barrier film by coating KCL nanocrystals and subsequently monitoring the ionic conductivity of the coated particles suspended in water. The primary interest in this process is the encapsulation and delayed release of solutes. The thickness of the shell is one of the independent variables by which we control the rate of release. It has a strong effect on the rate of release, which increases from a six-hour release (shell thickness is 20 nm) to a long-term release over 30 days (shell thickness is 95 nm). The release profile shows a characteristic behavior: a fast release (35% of the final materials) during the first five minutes after the beginning of the dissolution, and a slower release till all of the core materials come out.
Physics, Issue 66, Chemical Engineering, Plasma Physics, Plasma coating, Core-shell structure, Hollow particles, Permeability, nanoparticles, nanopowders
Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish
Institutions: Oregon Health and Sciences University.
Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo
preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo
imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.
Neuroscience, Issue 45, Zebrafish, synapse, electrophysiology, patch clamp, acetylcholine receptor, neuromuscular, cholinergic/action potential, myasthenic syndrome, motor control