Mikroakışkan kanalları döngüsel bir poliolefin (Zeonex ® 690R, Zeon Kimya A.Ş., Louisville, KY) imal edilir. Zeonex gözenekli Monolith oluşumunda kullanılan ışık yönettiği kimya için gerekli olan, 136 ° C cam geçiş sıcaklığı (T g) ve şeffaf UV. Mikro mikroakışkan platformu negatif (ters) özellikleri olan bir Nico electroformed ana-kalıp, mikro-sıcak kabartma tarafından imal edilir. Sıcak kabartma aşağıdaki şekilde yapılır: <ol><li> Kalıp ve polimer substrat paralel iki metal plaka arasına yerleştirilir. </li><li> Plakaları kabartma sıcaklık (166 ° C), Zeonex T g yukarıda 30 º C &#39;ye kadar ısıtılır. </li><li> Plakaları yaklaşık 5 dakika sonra bir araya sıkıştırılmaktadır ve basıncı 250 psi tutulur. </li><li> 1-2 dakika soğuması için izin verilen ısıtmalı plakaları, kalıp ve yüzey sonra kaldırılır ve daha sonra elle birbirinden ayrılır. </li><li> 1.5 mm çapında Wells örnek giriş ve toplama kanalları ucunda delinir. </li><li> Kabartmalı plastik substrat daha sonra termal Zeonex kapalı mikroakışkan kanalları oluşturmak için başka bir parça ile yapıştırılır. Kabartmalı substrat ve kapak parçası, UV-ozon veya plazma Sızdırmazlık verimliliğini artırmak ve kapalı kanal yapısı (Bhattacharyya ve Klapperich, 2007) sadakat sağlamak için termal yapıştırma önce tedavi edilebilir. </li></ol> Plastik mikroakışkan kanallar içinde katı faz ekstraksiyon (SPE) sütun Fabrikasyon Fabrikasyon katı-faz iki aşamalı bir süreçtir. İlk başta, fabrikasyon mikro SPE sütun yapışma plastik cihaz geliştirmek amacıyla yüzeyi modifiye edilmiştir. Yüzey fotopolimerizasyon yoluyla Aşılama polimerik mikro duvarları functionalize için kullanılır. Aşılama işlemi aşağıdaki gibi: <ol><li> Mikro,% 3 hidrojen soyutlayarak photoinitiator benzofenon, etilen diacrylate (EDA) ve metil metakrilat (MMA) 1:1 karışımı ile doldurulur. EDA, MMA ve benzofenon Sigma-Aldrich, St Louis, MO temin edilmektedir. </li><li> Çip ultraviyole maruz kalma aracı (CL-1000 UV Çapraz, UPV Inc, Upland, CA) 254 nm UV dalga boyu ve 200 mJ / cm 2 enerji az 10 dakika sonra UV ışınlanmış. Aşılama ikinci adımda Monolith hazırlanması için kullanılan polimerizasyon karışımı içinde dahil olsun yüzey gergin polimer zincirleri, H-soyutlama polimerizasyon ve sonuçları ile oluşur. </li><li> Fazla monomer 15 mcL vakum aspirasyon kullanarak metanol ile durulama kanalları kaldırılır. </li></ol> Mikrogözenekli bir polimer Monolith photografting işleminden sonra, mikro içinde oluşan silika partikülleri DNA ekstraksiyonu için tuzağa düşürürler. Gözenekli Monolith yerinde aşağıdaki gibi hazırlanmıştır: <ol><li> Yüzeyi modifiye kanal buma (% 15 ağırlık), EDMA (% 10 ağırlık), 1-dodecanol (% 52.5 wt), siklohekzanole (% 22.5 wt), DMPAP oluşan bir gökdelenin habercisi karışımı saygı ile (% 1 ağırlık ile dolu. monomerlerin) ve 0.7 mikron silika partikülleri (pre-polimer karışımı toplam hacmine göre% 15). Silika mikrosferler Polysciences, Inc (Warrington, PA) satın alındı ​​ve monomerler ve porogenic çözücüler Sigma-Aldrich, St Louis, MO temin edilmektedir. </li><li> Polimerizasyonu başlatmak için 1.1 dakika için 200 mJ / cm 2 çip UV çapraz bağlayıcı UV ile ışınlanmış ve fazla monomer vakum aspirasyon ile 15 mcL metanol ile durulama kanalları çıkarılır. </li><li> Daha sonra akışkan bağlantıları (NanoPorts TM bağlantıları, Upchurch Bilimsel, Oak Harbor, WA) mikroakışkan kanalların giriş ve toplama kuyuları bağlıdırlar . </li><li> Gözenekli Monolith sonra 100mL/hour bir akış hızında şırınga pompa kullanarak en az 30 dakika süreyle metanol ile tekrar yıkanır. </li></ol> Plastik mikroakışkan kanallar içinde CNT lizis Monolith Fabrikasyon CNT (karbon nanotüp) lizis Monolith Fabrikasyon iki aşamalı bir süreçtir. İlk olarak, fabrikasyon mikro plastik cihaz SPE sütunlar için aynı şekilde yüzey değiştirilmiş (aşılı). Photografting işleminden sonra, mikrogözenekli bir polimer monolit, çok duvarlı karbon nanotüpler ile emdirilmiş olduğu Mikrokanallı içinde oluşur. Gözenekli Monolith yerinde aşağıdaki gibi hazırlanmıştır: <ol><li> Çok duvarlı nanotüpler 2.27M bir konsantrasyon siklohekzanole yeniden bekletildi. Bu tabanda% 50 genlik az 30 dakika süreyle ultrasonicated ve sonifier hücre Topu (% 50 görev döngüsü Sonifier S-250Ultrasonik Branson, Danbury, CT) tarafından üretilen D,. Sonication CNTs etkili ve kararlı 0.5M süspansiyon sağladı. CNTs Nanolabs (Brighton, MA) satın alındı. </li><li> Yüzeyi modifiye kanalları şimdi siklohekzanole çözümü + CNTs kullanılır dışında benzer bir monolit öncü karışımı ile doldurulur. Monolith karışımı buma (% 15 ağırlık), EDMA (% 10 ağırlık), 1-dodecanol (% 52.5 wt), ve siklohekzanole + CNTs (% 22.5 ağırlıkça) oluşur. Bu çözüm için photoinitiator DMPAP (monomerler için saygı ile ağırlıkça% 1.13) eklenir. Monomerler, porogenic solventler ve DMPAPA Sigma-Aldrich, St Louis, MO temin edilmektedir. </li><li> Çip polimerizasyonu başlatmak için 0.9 dakika 120 mJ / cm 2 UV çapraz bağlayıcı UV ile ışınlanmış. Cihaz crossliker 180 derece saygısız ve 120 mJ / cm 2 az bir dakika süreyle 0.9 ışınlanmış. Fazla monomer 50 mcL vakum aspirasyon kullanarak metanol ile durulama kanalları kaldırılır. </li><li> Daha sonra akışkan bağlantıları (NanoPorts TM bağlantıları, Upchurch Bilimsel, Oak Harbor, WA) mikroakışkan kanalların giriş ve toplama kuyuları bağlıdırlar . </li></ol> Bakteriyel Numune Hazırlama CNT lizis Monolith ile kullanmak için Bakteriyel bir numune için numune hazırlama, numune kanal tıkanmaları önlemek için aşırı derecede konsantre değildir ki sağlamak için 600Nm bir optik yoğunluk ölçümü alarak içerir. <ol><li> Medya bakteri ile 600Nm bir OD okuma, bu biophotometer (Eppendorf BioPhotometer, Eppendorf Scientific, Inc, Westbury, NY) ile yapılır. OD okumaları aralığı 0,18-0,25 A. giren kadar örnek sulandırınız </li><li> 6500 rpm&#39;de 5 dakika santrifüj ve örnek süpernatantı süzün. </li></ol> Lizis <ol><li> GuSCN yeniden süspanse bakteri * +% 0,01 SDS +% 4 Proteinaz K (0.8mg/ml). Vortex kısaca (medya içinde bakteri 1 ml 1 ml GuSCN yeniden süspanse olması gerekir). Proteinaz K bakteriyel DNA eklenir ve DNAases ve RNAases inhibe proteinler denatüre yardım çözüm eklendi. Proteinaz K ek bir yararı, aynı zamanda, gram-negatif bakteriler genellikle daha fazla protein olmasına karşın, Gram-pozitif bakterilerin ve gram-negatif bakterilerin hücre duvarlarının büyük miktarlarda protein (içerdikleri için tercih edilir hücre duvarı proteinleri parçalayarak yardımcı .) Chaotropic tampon ayrıca deterjan hücre duvarı bozan proteinler denatüre proteinaz K yardımcı olur. Proteinaz K ve guanidinium tiyosiyanat (GuSCN) Qiagen A.Ş. (Valencia, CA) satın alındı. SDS (Sodyum dodesil Sülfat) Pierce (Rockford, IL) satın alınmıştır. </li><li> PEEK boru bağlı bir şırınga örnek hemen yükleyin. Herhangi bir hava sistemi böylece şırınga aspire edin. Nanoport ve kanal hortumu bağlayın. </li></ol> KDS100 şırınga pompası (KD Bilimsel, Holliston, MA tarafından üretilen) deneyleri için kullanılan ve debisi kullanılan tüm adımlar için 450 mcL / saat oldu. 450ul/hr örnek lizis kanal akışı. <ol><li> Örnek toplayın ve SPE ile DNA ekstraksiyonu (bkz. DNA ekstraksiyonu protokolü) devam edin. NOT: Bu bakteri örnek süspansiyon GuSCN olduğundan, Adım 2 Yük takip etmek gerek olmaz *. </li></ol> DNA Ekstraksiyon Protokolü Ekstraksiyon prosedürü 3 adımlardan oluşur: <ol><li> Yük. </li><li> Wash </li><li> Zehir. </li></ol> KDS100 şırınga pompası (KD Bilimsel, Holliston, MA tarafından üretilen) deneyleri için kullanılan ve kullanılan akış hızı 300 mcL tüm adımları / saat oldu. Yük <ol><li> Deney başlamadan önce 1-2 dakika süreyle chaotropic tamponu ile kanal (GuSCN * guanidinium tiyosiyanat) Durum. </li><li> 1:1 oranında chaotropic tamponu ile örnek karıştırın. </li><li> 3 dakika kanal üzerinden akış örnek karışımı. </li></ol> Yıkama <ol><li> Kanal 2 dakika süreyle% 70 etanol ile yıkayın. </li><li> Kanal 2 dakika süreyle% 100 etanol ile yıkayın. </li></ol> Zehir <ol><li> Millipore suda 3 dakika edilen DNA Zehir. 15 mcL, saflaştırılmış PCR hazır DNA toplayın. </li></ol> * GuSCN Qiagen kiti (Buffer RLT) kullanılmıştır.

<ol>
	<li>Bhattacharyya, A., Klapperich, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+and+chemical+analysis+of+plasma+and+ultraviolet-ozone+surface+treatments+for+thermal+bonding+of+polymeric+microfluidic+devices.">Mechanical and chemical analysis of plasma and ultraviolet-ozone surface treatments for thermal bonding of polymeric microfluidic devices.</a> Lab Chip. 7, 876-882 (2007).</li></ol>

The authors have nothing to disclose.

fabrication of the thermoplastic microfluidic channels

Laboratuvarda başarıyla doğrudan polimer / nanoparçacık kompozit microscale lizis ve katı faz ekstraksiyon sütunları kullanarak insan kan, idrar ve dışkı mikrolitrelik ve submicroliter birimleri nükleik asitlerin, izole var. Kurtarılan örnekleri herhangi bir ek temizleme olmadan, PCRable konsantre, küçük hacimli örnekleri vardır. Burada, nasıl sıcak kabartma ve ısı sızdırmazlık kullanarak termoplastik mikroakışkan cips imal göstermektedir. Sonra, kompozit katı faz sütunları oluşturmak için mühürlü çip aracılığıyla yerinde ışık aşılama yüzey ve polimerizasyon nasıl kullanılacağını göstermektedir. Biz aşılama ve bakteri hücre lizis için karbon nanotüp / polimer kompozit sütun polimerizasyonu göstermektedir. Daha sonra, silika / polimer kompozit sütun kullanarak çip üzerinde örnek nükleik asitlerin katı faz ekstraksiyon takip parçalama süreci göstermektedir. Ekli protokoller lizis ve katı faz ekstraksiyon sütunun her ikisinin de nasıl yapılacağı hakkında ayrıntılı talimatlar içerir.

Watch this Scientific Journal Video about Fabrication of the Thermoplastic Microfluidic Channels at JoVE.com

Fabrication of the Thermoplastic Microfluidic Channels

Burada sıcak kabartma ve ısı sızdırmazlık kullanarak termoplastik mikroakışkan cips nasıl imal göstermektedir. Sonra kompozit katı faz sütunları oluşturmak için mühürlü çip aracılığıyla yerinde ışık aşılama yüzey ve polimerizasyon nasıl kullanılacağını göstermektedir.

Termoplastik mikroakışkan Kanallar imalatı

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

fabrication-of-the-thermoplastic-microfluidic-channels

Research

JoVE Journal

Biology

13.3K Görüntüleme.  Boston University. Burada sıcak kabartma ve ısı sızdırmazlık kullanarak termoplastik mikroakışkan cips nasıl imal göstermektedir. Sonra kompozit katı faz sütunları oluşturmak için mühürlü çip aracılığıyla yerinde ışık aşılama yüzey ve polimerizasyon nasıl kullanılacağını göstermektedir.

Video: Fabrication of the Thermoplastic Microfluidic Channels

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Nöron Soma ve Aksonlar compartmentalization mikroakışkan Aygıt Fabrikasyon

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biyoloji

PDMS Device Fabrication and Surface Modification

PDMS Cihaz İmalat ve Yüzey Modifikasyonu

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

Ucuz mikroakışkan Cihazlar İmalat PDMS Non-plazma Yapışma

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

Tek kullanımlık Diagnostics mikroakışkan Uygulamaları

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Xenopus Oosit ifade İyon Kanalları Patch Kelepçe Kaydı

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Amperometrik Elektrotlar imalatı

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

MİYOJEN Engineered Doku oluşturur imalatı

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

Mikroakışkan Probe: Çalışma ve Yerelleştirilmiş Yüzey İşleme için kullanın

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

İyon Kanalları ve Fonksiyonel Analiz Mutagenez Heterologously Memeli Hücreleri cinsinden ifade edilir

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

GIRK Kanal Modülatörler belirlenmesi için Floresan Tarama Testi

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...