نشكر لوران Limozin، بيير ديلارد واستريد فاهل لمواصلة مناقشات مثمرة حول التطبيقات الخلوية. كما نشكر فريدريك البدو من مرفق غرف الأبحاث PLANETE لمساعدته مع الملاحظات SEM. وقد تم تمويل هذا العمل جزئيا من قبل مجلس البحوث الأوروبي عبر منحة رقم 307104 FP / 2007-2013 / ERC.

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الكتاب ليس لديهم ما يكشف.

ترتبط الخطوات الحاسمة ضمن بروتوكول المذكورة أعلاه إلى تشكيل البروتين نانو النقاط أو العودة ملء الفضاء حول النقاط التي كتبها طبقة ثنائية الدهون المعتمدة. الخطوة الأولى الحاسمة فيما يتعلق البروتين نانو النقاط هي إعداد حبة قناع. تنظيف غطاء الشريحة أمر بالغ الأهمية. وي...

<html> التصاق الخلايا تتم من خلال جزيئات متخصصة التصاق الخلية (الحدب)، البروتينات الموجودة على غشاء الخلية التي هي قادرة على الربط إلى نظيرتها في نسيج خارج الخلية أو على خلية أخرى. على خلايا الالتزام، ومعظم جزيئات الالتصاق بما في ذلك إنتغرين في كل مكان وكادهيرين، وتوجد في شكل مجموعات 1. تفاعل الخلايا الليمفاوية T (خلايا T) مع خلايا مقدمة للمستضد (ناقلات الجنود المدرعة) يوفر التوضيح ولا سيما ضرب من أهمية مجموعات مستقبلات شكلت في واجهة بين خليتين - غالبا ما يسمى المشبك المناعي. على تشكيل أول اتصال مع مستقبلات الخلايا APC، T (TCRs) على سطح T شكل خلية ميكرون مجموعات على نطاق والتي تكون بمثابة إشارات منصات 2، 3، 4، ومركزية في نهاية المطاف إلى تشكيل أكبر كتلة supramolecular المركزية (cSMAC )معشوقة = &quot;XREF&quot;&gt; 5، 6، 7. مؤخرا، تبين أن على الجانب APC، وتتركز في بروابط من TCR أيضا 8. في سياق T خلية APC التفاعل، ونشر الأنظمة الهجينة، حيث حاكت APC قبل العشب الصناعي functionalized مع البروتينات ذات الصلة، وقد تقدمت كثيرا فهمنا واجهة متشابك 2، 3، 4، 5، 6، 7 . في هذا السياق، هو ذات أهمية كبيرة لتصميم APC السطوح المحاكاة التي تستحوذ واحد أو أكثر من جوانب الخلية المستهدفة. على سبيل المثال، إذا تم المطعمة بروابط على طبقات ثنائية الدهون المعتمدة، فإنها يمكن أن تنتشر في الطائرة من طبقة ثنائية، تحاكي الوضع على سطح APC، وفي الوقت نفسه تسمح تشكيلcSMAC 6 و 7. وبالمثل، وقد حاكت مجموعات على APC من خلال خلق جزر بروابط في بحر من البوليمرات 9، 10، 11، 12، 13، 14. ومع ذلك، حتى الآن لم يتم الجمع بين هذه الميزات اثنين. نحن هنا وصف تقنية الرواية لخلق نانو النقاط مكافحة CD3 (الأجسام المضادة التي تستهدف مجمع TCR) وتحيط بها طبقة ثنائية الدهون مع الدهون نشرها. وتودع طبقة ثنائية باستخدام انجمير-بلودجيت / انجمير-شايفر تقنية 7 و 15 و 16 و إذا رغبت في ذلك، يمكن functionalized مع بروتين معين - على سبيل المثال، يجند للخلية إنتغرين T (وتسمى ICAM1). وبالإضافة إلى ذلك، ومكافحة CD3 النقاط البروتين فصول التوجيه الجامعييتم استبدال دينار مع الأجسام المضادة أو CAM آخر. في حين أننا اخترنا البروتينات لاستخدامها في المستقبل كمنصة لT الدراسات التصاق الخلية، واستراتيجية مفصلة هنا يمكن تكييفها لأي البروتين، وحتى الحمض النووي.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Glass coverslips</td>
			<td>Assistent, Karl Hecht KG&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Observation chamber</td>
			<td>Home made</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline surfactant concentrate (Hellmanex)</td>
			<td>Hella Analytics</td>
			<td>9-307-011-4-507</td>
		</tr>
		<tr>
			<td>Ultra-sonicator</td>
			<td>ThermoFisher</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Labbox</td>
			<td></td>
		</tr>
		<tr>
			<td>Crystallizer&#160;</td>
			<td>Shott</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidine</td>
			<td>Thermo Fischer Scientifique</td>
			<td>84607</td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Sigma-aldrich</td>
			<td>P3813</td>
		</tr>
		<tr>
			<td>Water MQ&#160;</td>
			<td>ELGA, Veolia France</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica beads</td>
			<td>Corpuscular Inc</td>
			<td>147114-10</td>
		</tr>
		<tr>
			<td>APTES</td>
			<td>Sigma-aldrich</td>
			<td>A3648</td>
		</tr>
		<tr>
			<td>BSA-Biotin</td>
			<td>Sigma-aldrich</td>
			<td>A8549</td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
		</tr>
		<tr>
			<td>Dansyl-PE</td>
			<td>Avanti Polar Lipids</td>
			<td>810330C</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-aldrich</td>
			<td>650471</td>
		</tr>
		<tr>
			<td>Gastight syringe&#160;</td>
			<td>Dominique Dustcher , France</td>
			<td>74453</td>
		</tr>
		<tr>
			<td>Film balance</td>
			<td>NIMA</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss, Germany</td>
			<td>TIRF-III system</td>
		</tr>
		<tr>
			<td>Aluminium Target&#160;</td>
			<td>Kurt J. Lesker Compagny, USA</td>
			<td></td>
		</tr>
		<tr>
			<td>Radio Frequency Magnetron sputtering Syst&#232;me&#160;</td>
			<td>modified SMC 600 tool by ALCATEL , France</td>
			<td></td>
		</tr>
	</tbody>
</table>

ligand nano-cluster arrays in a supported lipid bilayer

حاليا هناك اهتماما كبيرا في خلق صفائف أمر من الجزر البروتين لاصقة في بحر من سطح تخميلها للدراسات البيولوجية الخلية. في السنوات الماضية، فقد أصبح من الواضح بشكل متزايد أن الخلايا الحية تستجيب، وليس فقط لطبيعة البيوكيميائية من الجزيئات المقدمة لهم ولكن أيضا على الطريقة التي يتم بها عرض هذه الجزيئات. خلق البروتين أنماط الصغرى ولذلك المعيار الآن في العديد من المختبرات البيولوجية. متعدد الأنماط نانو هي أيضا أكثر سهولة. ومع ذلك، في سياق التفاعلات خلية خلية، هناك حاجة إلى نمط ليس فقط ولكن أيضا البروتينات طبقات ثنائية الدهون. وحتى الآن لم تكن هذه المزدوجة الزخرفة-PROTEO شحمي الوصول إليها بسهولة. نحن نقدم تقنية السطحية لخلق البروتين نانو النقاط معتمدة على الزجاج واقتراح طريقة لردم مساحة بين نقطة مع طبقة ثنائية الدهون المدعومة (SLB). من الصور تبييض التتبع الدهون الفلورسنت المدرجة في SLB، علينا أن نبرهن على طبقة ثنائية المعارض الكبيرة في وررسيولة انو. Functionalizing النقاط البروتين مع مجموعات الفلورسنت يسمح لنا صورة لها وتظهر أنها تنظم في شعرية سداسية منتظمة. النقطة حجم النموذجي هو حوالي 800 نانومتر، والتباعد تظاهر هنا هو 2 ميكرون. ويتوقع لهذه ركائز لخدمة منصات مفيدة لالتصاق الخلايا والهجرة والدراسات والميكانيكية الاستشعار عن بعد.

وقد تم تحليل الصور مضان لقياس المسافات وحجم النقاط. تم العثور على تباعد نموذجي ليكون 1900 ± 80 نانومتر، ونموذجي نقطة بحجم كان 600 ± 100 نانومتر (الشكل 1G). تم تعيين تباعد حسب حجم حبات تستخدم لقناع. يتم تعيين حجم نقطة من حجم حبة فضلا عن ظروف الترسيب. و?...

Watch this Scientific Journal Video about Ligand Nano-cluster Arrays in a Supported Lipid Bilayer at JoVE.com

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

نقدم بروتوكول لfunctionalize الزجاج مع بقع البروتين النانومترية تحيط بها طبقة ثنائية الدهن السائل. هذه ركائز متوافقة مع المجهر الضوئي المتقدمة، ومن المتوقع أن تكون بمثابة منصة للدراسات التصاق الخلايا والهجرة.

صالحة يجند نانو الكتلة في الدهن طبقة ثنائية المعتمدة

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

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6.9K Views.  CNRS, UMR 7325, CINaM.  نقدم بروتوكول لfunctionalize الزجاج مع بقع البروتين النانومترية تحيط بها طبقة ثنائية الدهن السائل. هذه ركائز متوافقة مع المجهر الضوئي المتقدمة، ومن المتوقع أن تكون بمثابة منصة للدراسات التصاق الخلايا والهجرة. 

Video: Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

إنشاء ثنائي الأبعاد ركائز منقوشة على الحبس البروتين والخلايا

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

الخلوية لاستخراج الدهن المستهدفة التخفيف النظائر المستقرة تحليل السائل الطيف اللوني القداس

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

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.

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.

ثابت الطريقة النتوء الضغط التي تسيطر عليها لإعداد الحويصلات الدهن نانو الحجم

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

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

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.

الدهون طبقة ثنائية الحويصلة انشاء طريق ميكروفلويديك النفث

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.

إعداد ميكا المدعومة الدهون طبقات ثنائية لارتفاع القرار المجهر الضوئي التصوير

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

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

قوة الذرية المجهر التصوير وقوة التحليل الطيفي لدعم الدهن طبقات ثنائية

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

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.

غشاء حيوي تلفيق من المذيبات بمساعدة الدهون طبقة ثنائية (SALB) طريقة

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

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

واحدة جزيء مضان المجهر على مستو المعتمدة طبقات ثنائية

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.