1. Preparing the lipids
<ol>
<li>In order to link proteins to the upper leaflet of a bilayer, we use Ni2+ chelating lipids, which anchor recombinant proteins with poly-histidine tags. Certain cell types may adhere non-specifically to Ni2+-coated bilayers, but T-cells generally do not, making this&nbsp;method optimal for studying T cell activation.</li>
<li>The lipid bilayer solution is made by preparing a solution of 20% Ni2+ chelating lipid and 80% phosphatidylcholine in the appropriate amounts in a glass tube with 1-2 ml of chloroform-methanol. <ol>
<li>Evaporate the solvent under a stream of nitrogen gas in a warm (30-37&deg;C) water bath. This usually takes around 15 minutes.</li>
<li>Remove any remaining solvent under high vacuum, using a lyophilizer for 90 minutes.</li>
</ol></li>
<li>Dissolve the lipids in 2% N-octyl-glucoside detergent in Tris-saline buffer, which&nbsp;creates a solution of mixed detergent/phospholipid micelles. The final phospholipid concentration should be 0.4 mM. To form liposomes, dialyze this solution against 3 changes of Tris-saline to remove detergent and form liposomes. We usually work with volumes on the order of 1 ml with 6 mm diameter Spectra/por #2 tubing with a cut off around 10 kDa. We are careful to exclude any air bubbles when clamping the tubing. We typically sterile-filter this solution and do the dialysis under clean conditions, handling it using 70% ethanol sterilized gloves.</li>
<li>This solution should be crystal clear. It is stored under argon gas to prevent oxidation. If the solution is turbid, then multi-walled vesicles have been&nbsp;generated, and you will need to start again.</li>
</ol>
2. Determining how much ICAM-1 and MHC are deposited on the planar bilayer
<ol>
<li>In order to set up the experiment, it is first necessary to determine how much ICAM-1 and MHC are deposited on a given surface area of Ni2+ chelating bilayer at a given concentration of soluble protein. To do this, deposit bilayers on 5 micrometer diameter glass beads, incubate the glass beads with protein solutions under conditions that approximate those in the flow cells used for imaging, and read out the density of bound protein by a flow immunofluorometric assay. </li>
<li>FITC-labeled antibodies with known numbers of fluorescein per molecule are used to detect the surface-bound proteins. Fluorescein standard beads are used to calibrate the flow microfluorimeter.</li>
<li>We will establish conditions that approximate those on antigen-presenting cells with 200 molecules/&mu;m2 of ICAM-1 and 0.2-20 molecules/&mu;m2 of I-Ek-MCC91-103 complex. </li>
</ol>
3. Cleaning the glass cover slips for forming planar bilayers
<ol>
<li>Planar bilayers form spontaneously when the liposomes fuse to glass, but only if the glass is properly cleaned.</li>
<li>When working with Piranha, we wear full protective clothing, including an acid apron and heavy, acid resistant gauntlets. Carefully segregate the&nbsp;waste from organic wastes and dispose of properly.</li>
<li>To prepare the acid Piranha solution, add 75 ml of concentrated sulfuric acid to a dry beaker, and carefully add 25 ml of 30% hydrogen peroxide. The solution rapidly heats up to greater than 100&deg;C.</li>
<li>Immerse the dry coverslips in the solution for 15 minutes. Remove from the solution and rinse in purified water for a minute on each side. Dry the coverslips using a vacuum source to drain off the purified water. Allow the Piranha solution to cool, and add to the Piranha waste container, which is left with the cap loose, well marked in the fume hood. When all of the liposomes, coverslips, and proteins are prepared, the immunological synapse is ready to be formed. </li>
</ol>
4. Forming the bilayers
<ol>
<li>To form the bilayers, our lab uses Bioptechs FCSII chambers, which have the advantages of integrated heating. The FCSII system is on a stainless steel base that clamps together a microfluidic manifold with a 0.5 mm top gasket, a microaqueduct slide coated with indium tin oxide for heating on one surface, with fluidic groove on the other side, a dust-free 0.25 mm gasket that defines the height of the flow chamber and the Piranha cleaned 40 mm round coverslip on which the bilayer is formed.</li>
<li>While the cover slip, upon which the bilayer is formed, needs to be highly hydrophilic and clean, the microacqueduct slide actually works better if it's more hydrophobic. Making the microaqueduct more hydrophobic is acheived by treating it with 1% HSA for 60 minutes at room temperature, then washing with pure water and drying completely after this conditioning process, as well as between uses. The coating of denatured proteins left by this procedure allows 1 &mu;l of liposome suspension to form a round, hemispherical drop that is &gt;0.25 mm high.</li>
<li>To assemble the flow cell and form planar bilayers, place the manifold, with the tubes directed upwards, upon a flat surface. Then, place the 0. 5 mm gasket with holes positioned over the fluidic tubes, making sure that it is seated flat. Gently place the microaqueduct side onto the gasket with the fluidic channels facing up, and make sure it seats flat without applying any downward pressure, until verifying that the holes in the slide align with the microfluidic tubes. Lay the dust-free 0.25 mm gasket with a rectangular cut out on top of the microaqueduct slide so that the channel is lined up with the fluidic channels and there are no major air spaces.</li>
<li>Place up to 5 x 1 &mu;l drops of liposome suspension on the surface of the microaqueduct slide in a pattern such that the center-to-center distance between each drop is 2.5 mm. The 5 liposome drops may have different compositions, but in this case we will only form two bilayers, one with no Ni2++ chelating lipids and one with 10% Ni2++ chelating lipids.</li>
<li>Once these drops are in place, carefully place the cover slip down on the surface in one motion. Clamp onto the base as quickly as possible. The liposome drops should make contact with the cover slip. This contact should not be broken, since the bilayers have probably already formed; any disruptions may result in defective bilayers.</li>
<li>It is important to remember to mark the positions of the bilayers with a few small ink spots on the microacqueduct slide to aid positioning of the coverslip on the microscope. Wait 20 minutes to make sure the system has equilibrated. Transfer all the solutions using a 20 ml syringe connected to about 20 cm of flexible tubing and a 3-way stopcock. Connect another port of the stopcock to the flow cell by a short length of tubing to minimize the dead volume between the stopcock and the flow cell. Fill the syringe with HEPES-buffered saline containing 2 mM MgCl2, 1 mM CaCl2, 5 mM glucose and 1% human serum albumin.</li>
<li>Prime the tubing and stopcock so there are no air bubbles. Connect this to the flow cells and push through 5 ml in one motion while watching to make sure that no air bubbles pass over the bilayer. Now, you will have your bilayers.</li>
</ol>
This video article supports "Hunter to Gatherer and Back: Immunological Synapses and Kinapses as Variations on the Theme of Amoeboid Locomotion" by <a href="http://arjournals.annualreviews.org/action/doSearch?action=runSearch&amp;type=advanced&amp;result=true&amp;prevSearch=%2Bauthorsfield%3A%28Dustin%2C+Michael+L.%29" target="_blank">Michael L. Dustin</a>, <a href="http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.cellbio.24.110707.175226">Annual Review of Cell and Developmental Biology</a>, Volume 24, 2008.

supported planar bilayers for the formation of study of immunological synapses and kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Watch this Scientific Journal Video about Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse at JoVE.com

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

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12.5K Views.  New York University - NYU. Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy. 

Video: Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Investigating the Immunological Mechanisms Underlying Organ Transplant Rejection

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...