Bu çalışma NIH araştırma fonu (DK090868 ve TI GM092930) tarafından desteklenmiştir. Kafesli bir rapamisin analog bir patent var.

1. Plazmid DNA transfeksiyon <ol><li> 37.5 ul dH 2 O kadar floresan proteini tagged: plazmid DNA&#39;lar, 0.5 mikrogram membran-gergin FRB (bundan sonra LDR olarak anılacaktır) ve 0.5 mg FKBP-Tiam1 (Rac aktivatör T-hücreli lenfoma invazyon ve metastaz uyaran protein 1) ekle 1 ul FuGENE HD ekleyin. </li><li> 20 dakika için oda sıcaklığında vorteks ile inkübe. Bu arada, adım 1,3-1,8 geçin. </li><li> 50 ul poli-D-Lizin içeren bir 8-de haznesinin her oyuğa yıkanır. </li><li> DH 2 O her iyi merkezi yıkayın </li><li> % 80-85 konfluent NIH-3T3 hücreleri Trypsinize ve 10 ml kültür ortamı (% 10 FBS DMEM) ye tamamlanır. </li><li> Süspansiyon 375 ul çıkarın ve 3 dakika 1750 rpm&#39;de santrifüj. </li><li> Pelet rahatsız dikkat ederek, medya aspire. </li><li> DMEM w /% 10 FBS ve Pastör pipetiyle 750 ul ekleyin. </li><li> DH 2 O / DNA / FuGENE HD çözümü için hücre süspansiyonu ekleyin ve hafifçe karıştırın. </li><li> 250 ekleHer bir kuyuya hücre / DNA süspansiyonu ul (3 kuyu kadar dolduracaktır). </li><li> 37 Transfekte hücreler inkübe ° C ve 15-18 saat boyunca% 5 CO2. </li></ol> 2. KİB-Bir Çözüm hazırlanması <ol><li> Sentezlemek kafesli rapamisin-biotin adduct (KİB). Ayrıntılar için &quot;KİB Sentetik düzeni&quot; bakınız. Kısaca, kafes ve biyotin yarımlar ayrı olarak hazırlanır. Kafes yarımı sonra rapamisinin (LC laboratuar) ile konjuge olup, ürün daha sonra CRB verecek şekilde tıklama reaksiyonu içinde bir 7 vasıtası ile biyotin yarımına bağlanmıştır. </li><li> DMSO içinde CRB bir 1 uM stok çözeltisi hazırlayın. </li><li> 0,5 ml Eppendorf tüp PBS 250 ul olarak 1mg avidin eritin. </li><li> Çözünmüş avidin için CRB stok solüsyonundan 1 ul ekleyin. Ters tarafından bir kaç kez karıştırın. </li><li> CRB-avidin konjugatı (CRB-A verecek şekilde 15 dakika oda sıcaklığında inkübe) Çözümü. </li><li> Ilişkisiz küçük moleküller uzaklaştırın ve saflaştırmak KİB-A boyutu-dışlama sütunu kullanarak. </li></ol> 3. Mikroskopi ve Subselüler Düzeyinde Işık Aydınlatma <ol><li> Serum 12 saat boyunca FBS olmadan DMEM içinde transfekte edilmiş hücrelerin açlık. </li><li> Yavaşça hücrelerini medya çekilerek hazırlanmış 400 nM KİB-bir çözüm (adım 2.6 dan itibaren) 250 ul ekleyin. </li><li> Işık tedavisi öncesi ve sonrası Subselüler çözünürlükte canlı hücreler protein dinamikleri görselleştirmek için, iplik diski mikroskobu (Şekil 2a) kullanıldı. UV aydınlatma epi-floresan ışığı (Şekil 2b) ile UV LED ışık (365 nm) için optik bağlantı sağlandı. </li><li> Metamorph yazılımı Transfekte edilmiş hücrelerin floresans görüntüler, her 15 saniye yakalamak için kullanıldı. </li><li> UV ışığı ile heyecanlandım floresan boya ile kaplanmış bir cam levha kullanılarak bir aydınlatma noktanın koordinatları ve boyutu tanımlayın. Boyutu olabilirfarklı büyütmeler objektif lens uygulayarak ve farklı çekirdek boyutu ile optik fiberler seçilerek çeşitlendirilebilir. </li><li> Hücre periferinde membran ruffling, saçmak UV ışık arka seviyelerini belirledikten sonra. Aşağıdaki iki adım lokalize etkileri önceki karşılaştırmak vardır. </li><li> Küresel bir cıvalı lamba tarafından sağlanan 365 nm ışık hücreleri ışın tedavisi. </li><li> , Uncaged DMSO içinde ve sonra PBS (400 nM final) içinde eritilir ve böylece orijinal rapamisin ve küresel etkilere sebep olmak için görüntüleme odasına solüsyonu ilave edin. </li></ol> 4. Temsilcisi Sonuçlar Temsili sonuçlar bir örneği, Şekil 3 &#39;de gösterilmiştir. Bir hücresel kenarında küçük bir bölgede UV ışık ışınlama hızla lokalize oluşumu uyarılan ışınlanmış alanında ve yakın sadece katlar. Bu WHI iki, biz o zaman küresel UV ile ışınlanmış hücreleri, aslında yerel etkinlik, ya da toplu medya rapamisin eklendiğini onaylamak içinch membran boyunca küresel fırfır oluşumuna bağlı. Bir kantitatif analiz için, dört parçaya bölünür hedef hücrelere bölünmüş ve fırfır yerel üzerine her çeyrekte oluşumu gibi denetimleri (Şekil 4) dahil olmak üzere, çeşitli koşullar altında küresel uyarılmasını takiben sıklığı sayılır. Sadece bu UV ışınlama kombinasyonu, KİB-A, ve yerel ruffling indükler uygun FKBP-FRB yapıları olduğunu. <img alt="Şekil 1" src="/files/ftp_upload/3794/3794fig1.jpg" /> Şekil 1. Kafesli rapamisin-avidin konjuge (KİB-A) ve UV ışık kullanılarak mekansal sınırlı protein Dimerleşme şematik. UV ışını kimyasal dimerizer (HE-Rapa, C40-O-(2-hidroksietil) rapamisin) ve yan-ürünün yayın ile sonuçlanan, rapamisin ve biotin ile linker parçalayarak. Serbest dimerizer sonra hücrelerin içine yayılır ve FKBP-POI (ilgi protein) ve plazma memb arasındaki dimerizasyon indüklerRane sadece ışınlanmış bölgesi (mavi daire) yakınında FRB demirledi. UV ışını olmadan, FKBP ve FRB dolayısıyla hiçbir etkisi üreten, (kırmızı çarpı) doçent yok. (Copyright @ ACS2011) 6 <img alt="Şekil 2" src="/files/ftp_upload/3794/3794fig2.jpg" /> Şekil 2. Özelleştirilmiş konfokal floresan mikroskop resmi. Konfokal mikroskop (a) önden görünüşü (Axiovert 200, Zeiss ters). EPI, floresans ve UV aydınlatma için bir kaplin ünitesi (b), Yakın çekim, üstten görünüşü. <img alt="Şekil 3" src="/files/ftp_upload/3794/3794fig3.jpg" /> Şekil lokalize UV ışını ile indüklenen bir transfekte NIH3T3 hücrede 3. Mekansal sınırlı fırfır oluşumu. YFP etiketli FKBP-Tiam1 (Rac aktivatörü) ve LDR (FRB membran-demirlemiş) ile transfekte bir NIH3T3 fibroblast hücre Konfokal floresan görüntüleri L ile kenarına yakın bir sınırlı alanda ışınlama yapıldı15 sn toplu medyada KİB-A konjugat varlığında 210 sn &#39;den için 365 nm&#39;de ED ışık. Bu tedavi 1560 sn rapamisin son 400 nM 1063 sn ve küresel Ayrıca az 1 saniye civa lambası ile global aydınlatma izledi. Hücre başında (a) neredeyse hiç ruffling faaliyet gösterdi ancak gösterdi uzaysal lokalize aydınlatma sonra ruffling sınırlı (sarı daire) (b). Hücre yerel fırfır oluşumu Rac olarak uzaysal sınırlı aktivasyonu sonucu olduğunu gösteren, global aydınlatma ve rapamisin (cd) ilavesinden sonra küresel fırfır oluşumu gösterdi. Ölçek çubuğu: 10 mikron. (Copyright @ ACS2011) 6 <img alt="Şekil 4" src="/files/ftp_upload/3794/3794fig4.jpg" /> Şekil 4. Transfekte NIH3T3 hücrelerinin kadranda fırfır oluşumu sıklığı. CRB-A ve aşağıdaki genel UV ışını 13 hücreleri üzerinde yapıldı. CRB-A ile lokal UV ışını </s&gt; Ayrıca (UV radyasyon) Lüks, avidin olan veya olmayan yerel UV ışınlaması sırasıyla 13, 5 ve 6 hücreleri üzerinde uygulanmıştır. Gözlenen hücrelerin hiçbiri UV radyasyon, veya veya toplu ortamda avidin olmadan yerel UV ışınlaması olmadan KİB-A ile fırfır oluşumu gösterdi. Düşük arka fırfır oluşumu ile hücrelerde verileri toplanmıştır ancak bu rapamisinin (nihai 400 nM) ilavesi sonucu genel oluşması fırfır teyit tarafından her bir deney sonra kontrol rapamisin indüklenen fırfır oluşumu, bir kapasiteye sahip olan. Bu sonuçlar, CRB-A ve yerel UV ışını hücrelerinde yerel fırfır oluşumu için gerekli olduğunu gösterdi. (Copyright @ ACS2011) 6 Kısaltmalar: FKBP: FK506-bağlayıcı protein FRB: FKBP-rapamisin-bağlayıcı protein Tiam1: T-hücreli lenfoma invazyon ve metastaz uyaran protein 1 (Rac GEF) LDR: Lyn-DSAG linker-FRB (Lyn: Lyn kinaz N-terminal 11 amino asitleri) HE-Rapa: C40-O - (2-hidroksietil) rapamisin KİB: biotin grubu ile kafesli rapamisin KİB-A: avidin için CRB konjuge

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Çıkar çatışması ilan etti.

Biz saniyelik bir zaman ölçeği üzerinde doğru bir hücre içi yerde Rho GTPaz aktivitesi işlemek üzere FKBP-FRB heterodimerization sistemine sahip bir yeni bileşik kafeste birlikte çalıştığı bir yöntem tarif. Bu yaklaşımın üç sınırlamalar vardır. Metodu plazma membranında dimerizer difüzyonu önlenmesi veya izin bağlı olduğu Birincisi, hedef hücresel plazma membranında konuma yada çevresi olması gerekmektedir. KİB kimyasal yapısı daha ileri optimizasyon bil...

<table border="1"><tbody><tr><td> Reaktif / ekipman Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Rapamisin </td><td> Tecoland </td><td> RAPA99 </td></tr><tr><td> FuGENE HD </td><td> Roche </td><td> 04709691001 </td></tr><tr><td> Avidin </td><td> Sigma </td><td> A9275-10mg </td></tr><tr><td> DMSO </td><td> Sigma </td><td> D2650-5X5ML </td></tr><tr><td> Boyut-dışlama sütun </td><td> GE Healthcare </td><td> Spin Tuzak G-25 </td></tr><tr><td> Cam alt 8-iyi kamara </td><td> Thermo Scientific </td><td> 12-565-47 </td></tr><tr><td> Ters Floresan mikroskop </td><td> Zeiss </td><td> Axiovert200M </td></tr><tr><td> UV ışık kaynağı LED </td><td> Rapp Opto Elektronik </td><td> UVILED </td></tr><tr><td> Konfokal dönen disk </td><td> Yokogawa Electric Corp </td><td> CSU10 </td></tr><tr><td> CCD kamera </td><td> Photonics </td><td> ORCA-ER </td></tr><tr><td> 100x Objektif lens </td><td> Zeiss </td><td> Planı Apochromat </td></tr></tbody></table> KİB Sentetik şeması (Copyright ACS2011) 6 <a href="/files/ftp_upload/3794/3794scheme1_lg.jpg"><img src="/files/ftp_upload/3794/3794scheme1.jpg" alt="Şema 1" /></a> Sentetik koşullar: (a) BnBr, K 2 CO 3, DMF (b) 3 f.HNO, AcOH (c), TFA (d) propargil-Br, K 2 CO 3, DMF, (e) gliserol, kedi. PTSA, Toluen (f) NaBH3CN, TiCl 4, MeCN (g) Tf 2 O, Piridin (h) metanol HCl, (i) LiAlH 4, THF (j) TsCl, Piridin, CH 2 Cl 2 (k) NaN 3, DMF (l) 8, 2,6-di-t-butylpyridine, CH 2 Cl 2 (m) 13, CuSO 4, askorbat, 2-propanol, 2 H, O, CH2 Cl2.

spatio-temporal manipulation of small gtpase activity at subcellular level and on timescale of seconds in living cells

Büyük zamanmekansal hassasiyetle küçük guanozin triphosphatases (GTPases) ve Rho ailesinin Dinamik yönetmelik çeşitli hücresel fonksiyonları ve olaylar 1, 2 için gereklidir. Onların spatiotemporally dinamik yapısı gerçek zamanlı 3 de, faaliyet ve yerelleşme görselleştirme tarafından ortaya konmuştur. Moleküler düzeyde çeşitli hücresel fonksiyonların rolleri daha derin bir anlayış kazanmak için, bir sonraki adım, kesin Subselüler konum ve zamanlama ile protein faaliyetlerinin pertürbasyon olmalıdır. (1) rapamisin bağlı FKBP-FRB heterodimerization ve rapamisin (2) Fotoğraf-kafes yöntemi: Bu hedefe ulaşmak için, ışık kaynaklı, iki tekniği birleştirerek küçük GTPases bir uzay-zamansal olarak kontrollü aktivasyon için bir yöntem geliştirdik. Rapamisin aracılı FKBP-FRB heterodimerization kullanımı ile, hızla küçük GTPases includi aktivasyonu veya inaktivasyonu indüklenebilir için bir yöntem geliştirding Rac 4, rapamisin FRB tutturulduğu plazma zarına, FKBP-erimiş GTPases, ya da bunların aktivatörlerinin translokasyonu indüklemektedir hangi Cdc42 4, RhoA 4 ve 5 Ras,. Bu heterodimerization sistemi ile bağlantı için, biz de rapamisin analoglarının bir foto-kafes sistemi geliştirdik. A foto-kafeste bileşiğin aktivitesi olan bir kafes grubu olarak bilinen bir photocleavable koruyucu grup ile bastırılmış, küçük bir moleküldür. Tamamen heterodimerization faaliyetleri bastırmak için, hücrelerin 6 içinde kimyasal bir dimerizer olarak neredeyse hiçbir plan aktivitesi yol, elde edilen büyük ve karmaşık bir plazma zarı geçemez öyle bir makromolekül tethered bir kafese rapamisin tasarlanmıştır. Şekil 1 bizim bir düzeni göstermektedir sistem. Bu iki sistemin kombinasyonu ile, yerel saniyelik bir zaman ölçeği üzerinde plazma membranı bir Rac aktivatörü işe ve Subselüler lev ışık kaynaklı Rac aktivasyon eldeEl 6.

Watch this Scientific Journal Video about Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells at JoVE.com

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Işık tarafından küçük GTPaz aktivitesinin yerleşim-zaman kontrolü için bir yöntem tarif edilmiştir. Bu yöntem, rapamisin indüklenen FKBP-FRB heterodimerization ve foto-kafes sistemleri dayanmaktadır. Işık ışınlama Optimizasyonu Subselüler düzeyindeki küçük GTPases bir uzay-zamansal olarak kontrollü aktivasyon sağlar.

Yaşayan hücreler içinde Subselüler Düzeyinde ve Seconds Zaman ölçeği üzerinde Küçük GTPaz Faaliyet uzay-zamansal Manipülasyon

Dynamic regulation of the Rho family of small guanosine triphosphatases (GTPases) with great spatiotemporal precision is essential for various cellular ...

spatio-temporal-manipulation-small-gtpase-activity-at-subcellular

Research

JoVE Journal

Bioengineering

10.8K Görüntüleme.  Johns Hopkins University. Işık tarafından küçük GTPaz aktivitesinin yerleşim-zaman kontrolü için bir yöntem tarif edilmiştir. Bu yöntem, rapamisin indüklenen FKBP-FRB heterodimerization ve foto-kafes sistemleri dayanmaktadır. Işık ışınlama Optimizasyonu Subselüler düzeyindeki küçük GTPases bir uzay-zamansal olarak kontrollü aktivasyon sağlar.

Video: Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Yaşayan Hücreler Moleküler Rotorlar arasında Flüoresans Ömrü Görüntüleme

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Biyomühendislik

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Tek Adacık Düzeyde Oksijen mikroçevresinin Nicel ve Geçici Kontrol

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cerrahi Dokular İlköğretim İnsan Kolon Tümör Hücreleri İzolasyon ve Çekiş Sitometrinin Yumuşak Elastik Yüzeyler Doğrudan onları Kültürleme

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Tek Hücre Salgıları uzay-zamansal Görüntüleme için bir etiket içermeyen Tekniği

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Konjuge Polimerler tarafından Hücreler Elektrik Etkinliği Yaşam Optik Kontrol

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

içinde Microengineered İnsan Bağırsak Villi Oturma mikrobiyomu Co-kültür, bir Gut-on-a-Chip mikroakışkan Aygıt

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Yalıtım insan alveoler kapaklarini ve Osteojenik etkinlik kapaklarini türetilmiş hücre D vitamini etkileri Mezenkimal kök hücrelerin

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Yamultma stres gelişmekte olan zebra balığı kalp oransal etkileri 4 boyutlu görüntülerini yakalamak için ışık sayfalık floresans mikroskobu

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called mechanotransduction. A crucial step in mechanotransduction is the transmission of forces between the external and internal environments. To transmit forces, there must be a sustained, unbroken physical linkage created by a series of protein-protein interactions. For a given protein-protein interaction, mechanical load can either have no effect on the interaction, lead to faster disassociation of the interaction, or even stabilize the interaction. Understanding how molecular load dictates protein turnover in living cells can provide valuable information about the mechanical state of a protein, in turn elucidating its role in mechanotransduction. Existing techniques for measuring force-sensitive protein dynamics either lack direct measurements of protein load or rely on the measurements performed outside of the cellular context. Here, we describe a protocol for the F&#246;rster resonance energy transfer-fluorescence recovery after photobleaching (FRET-FRAP) technique, which enables the measurement of force-sensitive protein dynamics within living cells. This technique is potentially applicable to any FRET-based tension sensor, facilitating the study of force-sensitive protein dynamics in variety of subcellular structures and in different cell types.

Here, we present a protocol for the simultaneous use of F&#246;rster resonance energy transfer-based tension sensors to measure protein load and fluorescence recovery after photobleaching to measure protein dynamics enabling the measurement of force-sensitive protein dynamics within living cells.

Kuvvet duyarlı Protein Dynamics floresan teknikleri bir arada kullanarak canlı hücrelerdeki ölçümü

Cells sense and respond to physical cues in their environment by converting mechanical stimuli into biochemically-detectable signals in a process called ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Doku Seviyesi Mekaniğinin Kontrol Altına Almak İçin Uyumlu Yüzeylerde İnsan Embriyonik Kök Hücre Kolonilerinin Geometrisinin Desenlemi

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...