Biz ATP deneyinde reaktiflerin hacimleri üzerindeki tasarruf fikri Juliann Jaumotte kabul. Biz bu çalışmalar için mali destek sağlayan Mary Caruso, Deb Willson ve Jackie Farrer ve Eczacılık Mylan Okulu muhteşem idari destek için gönülden teşekkür ederiz. Teşekkür de Hunkele korkunç Hastalıkları Vakfı ve Parkinson ve Hareket Bozuklukları birincil nöronal çalışmaların mali destek Vakfı kaynaklanmaktadır.

Yazarların hiçbiri ifşa herhangi çatışmalar var.

Tüm üç canlılığı deneylerde sinyal gücü, doğrusal ve kaplama yoğunluğu ile ilişkili olduğunu bulmuşlardır. Ancak, tüm deneyler, 2-kat veya kaplama yoğunluğu 1.5 kat değişikliklere karşı eşit ölçüde hassastır. N2a hücreleri için, kızılötesi deneyler özellikle düşük kaplama yoğunluklarında, ATP deneyi daha az duyarlıdır. Kızılötesi deneyler ATP daha az duyarlı olan, ancak DRAQ5 + Safir deneyler ve α-tubulin deneyleri ki bunlar, N-asetil sistein ve yüksek ölçüde koruyucu bi...

Biyolojik bilimlerde en yaygın canlılığı tahlil hücre sayıları içerir. Bu 2013/04/29 ve 2013/04/30 üzerinde anahtar kelime ya &quot;in vitro&quot; veya &quot;kültür&quot; ile PubMed&#39;de çıktı üst (en son) 200 yayınların analizi ile kanıtlanır. Bu yayınların, manuel hücre sayısı sayımı dahil olmak üzere% 23.5 kullanılan hücre sayımı tahlilleri, otomatik hücre sayısı görüntüleme yazılımı ile sayar ve Trypan mavi dışlama. Canlı / Ölü deney, bu yayınların% 1 kullanılmıştır. MTT (3 - (4,5-dimetiltiazol-2-il) -2,5-difeniltetrazolyum bromür) kullanılarak yayın sayısı metabolik canlılığı için tahlil% 11 idi. Edebiyat Bu anket aynı zamanda hücre sayımı tahlilleri ile birlikte böyle MTT gibi analizleri kullanılarak yayın sayısı sadece% 3.5 olduğunu göstermektedir. Hücre sayısı ile birlikte hücresel fonksiyonunun değerlendirilmesinde, tek başına bir canlılık deneyi kullanmak için eğilim hücresel i değerlendirmek için iyi bir seçim gibi görünüyor rağmenntegrity. Geriye kalan hücreler fonksiyonel ya da 1,2 kuyu içinde mevcut olmasına rağmen sağlıklı olmayabilir, çünkü kendi başına hücre sayımları yeterli değildir. Bunun tersine, ATP gibi fonksiyonel önlemler artırmak veya hücre sayısındaki paralel değişikliklerle yokluğunda azaltabilir. Hücre sayısı metabolik okumalanna ayrılması ATP ve MTT testleri tek canlılık deneyi olarak asla kullanılmamalıdır gerektiğini göstermektedir. Bu raporda, hücresel yapılar ve metabolik fonksiyonu hem de anket üç canlılığı deneyleri gelemez kendisi tarafından herhangi bir tahlil daha hücresel bütünlüğü daha kapsamlı bir görünüm için, tarif edilmektedir. Bizim deneylerinin iki 700 ve 800 nm kanallarda floresan ölçen bir enfraruj görüntüleyici gerektirir. Gürültü daha yüksek sinyal-gürültü oranı 3 yol açan, kızılötesi dalga boyu düşüktür. Kullandığımız Odyssey kamera, 4.5 günlük dinamik aralık ve 16, TRANSLATIN bir bit derinliğe sahipkızılötesi 2 16 veya 65.536 tonları g. Bu sadece her dalga boyu için renk 2 8 veya 256 tonunu tanıyor 8-bit renk görüntüleme, tezat olabilir. Böylece, 16-bit görüntüleme ince çözünürlüğe sahiptir. Bu orijinal kızılötesi genellikle görsel sunumu için yayınlanmış olan raporlarda (800 nm) ve kırmızı (700 nm), yeşil yalancı olduğu not edilmelidir. Odyssey kameralar genellikle Western blot ve In-Hücre Batılıların 4-7 hem de kullanılmaktadır. In-Cell formaldehit sabit hücreleri üzerinde Western ilgi duyulan herhangi bir protein karşı birincil antikor kullanımı ve kızıl ötesi floresan ikincil antikorlarla da etiketleyin. Bu teknik, fosforilasyon uç noktalar 6 için özellikle yararlı olduğu da bilinmektedir. Bizim In-Cell Western olarak, sitoskeletal proteinler α-tübülin ya da 800 nm kanalda nöronal mikrotübül ilişkili protein 2 (MAP2) için sabit hücreleri boyamak. Bu proteinler yüksek sinyal-gürültü oranı elde etmek için yeteri kadar bol miktarda bulunmaktadır. Biz de 700 yılında bizim plakaları lekeDRAQ5 leke ile ve safir leke ile sitoplazmada için çekirdekleri için nm kanalı. Sitoskeletal proteinler ve DRAQ5 + Sapphire lekeleri Hem böylece hücresel yapılarını yansıtmaktadır. Üçüncü canlılığı deneyi, metabolik fonksiyonu büyüklüğüne ve adı &quot;Hücre Titer Glo.&quot;, Bu lusiferaz bazlı tahlilde, parlaklık değerleri ATP seviyeleri doğrudan orantılıdır. ATP tahliller genel olarak canlı hücreler 8-12 ölçmek için kullanılır. Hücre başına ATP çıkış toksin tedaviler bir fonksiyonu olarak değişebilir Ancak, tahlilin adına kelime &quot;titre&quot; da dahil olmak üzere bir yanlış isim biraz ve hücre sayısı 8 ile orantılı olarak, bu nedenle her zaman değil. ATP düzeyleri de sirkadyan ritim 13 ve 14, hücre bölünmesi ve hücre farklılaşması 15 etkilenebilir. ATP metabolik sağlam bir ölçüsüdür çünkü Bununla birlikte, burada gösterilen ATP deneyi gerçekleştirmek için basit ve faydalıdırcanlılığı 16-21, başına numara hücre değilse. Batılıların bu nedenle tek başına herhangi bir deneyde daha hücresel bütünlüğün daha kapsamlı bir resmini verir In-Cell kızılötesi tamamlamak için bu tahlil kullanma.

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Cell Titer Glo</td>
			<td style="width:112px;">Promega</td>
			<td style="width:93px;">G7572</td>
			<td style="width:507px;">Buy in 100 ml quantities and aliquot, instead of purchasing the more expensive 10 ml quantity. Reconstituted, unused reagents can be refrozen at -20 &#176;C for at least 21 weeks</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">18% Formalin</td>
			<td style="width:112px;">Thermo-Shandon</td>
			<td style="width:93px;">9990244</td>
			<td style="width:507px;">Buying this fixative avoids the weighing out of formaldehyde powders and boiling of the solution; exposure to vapors is thereby minimized</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sucrose</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">S0389</td>
			<td style="width:507px;">It is not essential to add this to formaldehyde solutions but it improves the appearance of the fixed cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Odyssey Block</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">927-40003</td>
			<td style="width:507px;">This fish serum can be bought in bulk and frozen at -20 &#176;C for long term use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Triton-X 100</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">21568</td>
			<td style="width:507px;">We store a stock solution of 10% Triton-X 100 in sterile water at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Monobasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S468</td>
			<td style="width:507px;">One can also buy PBS tablets or 10x PBS solutions, but they are more expensive</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Phosphate Dibasic</td>
			<td style="width:112px;">Fisher</td>
			<td style="width:93px;">S373</td>
			<td style="width:507px;">See above</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sodium Azide (250x)</td>
			<td style="width:112px;">Ricca Chemical Company</td>
			<td style="width:93px;">7144.8-16</td>
			<td style="width:507px;">Do not buy the powder because sodium azide is very toxic. We store all our used antibodies in 1x sodium azide at 4 &#176;C until they become contaminated with debris</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-&#945;-tubulin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">T5168</td>
			<td style="width:507px;">This antibody is expensive but can be greatly diluted and is highly specific</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Mouse anti-MAP2</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:93px;">M9942</td>
			<td style="width:507px;">This antibody is expensive but is highly specific (a prerequisite for In-Cell Westerns)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">800 nm Goat anti-mouse IgG</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">926-32210</td>
			<td style="width:507px;">Other companies also sell infrared secondary antibodies. Be sure to purchase the highly cross-adsorbed antibodies and note that concentrations of IgGs may vary with the source</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">DRAQ5</td>
			<td style="width:112px;">Biostatus</td>
			<td style="width:93px;">DR50200</td>
			<td style="width:507px;">This compound used to be sold by LI-COR at 1 mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:109px;">Sapphire</td>
			<td style="width:112px;">LI-COR</td>
			<td style="width:93px;">928-40022</td>
			<td style="width:507px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Luminometer</td>
			<td style="width:129px;">PerkinElmer</td>
			<td style="width:189px;">VICTOR3 1420 multilabel counter</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Odyssey Imager</td>
			<td style="width:129px;">LI-COR</td>
			<td style="width:189px;">9201-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:196px;">Shaker/Mixer</td>
			<td style="width:129px;">Research Products International</td>
			<td style="width:189px;">248555</td>
			<td></td>
		</tr>
	</tbody>
</table>

viability assays for cells in culture

Bir mikroskop Manual hücre sayımları hücre canlılığının değerlendirilmesinde hassas bir yoludur, ancak zaman alıcı ve bu nedenle pahalıdır. Bilgisayarlı canlılığı deneyleri ekipman açısından pahalı ama daha hızlı ve daha objektif manuel hücre sayımları daha olabilir. Bu rapor, bu tür üç canlılığı tahlillerin kullanımını tarif eder. Bu tahlillerin ikisi kızılötesi ve bir parlak olan. Her iki kızılötesi tahlilleri bir 16 bit Odyssey Imager güveniyor. Bir kızılötesi deney sitosol için safir leke ile kombine çekirdekleri için DRAQ5 leke kullanır ve 700 nm kanalında görüntülenmiştir. Diğer kızılötesi deney, bir In-Cell Western, sitoskeletal proteinler (α-tubulin veya mikrotübül ilişkili protein 2) karşı antikorlar kullanır ve 800 nm kanal bunları etiketler. Üçüncü canlılık deneyi ATP için yaygın olarak kullanılan bir ışıldayan deney, ama biz maliyet tasarruf için önerilen hacminin dörtte kullanın. Bu ölçümler her doğrusaldır ve ce sayısı ile ilişkiliLLS kaplama, ancak duyarlılık değişir. Tüm deneyler üç zaman alıcı mikroskobu aşmak ve tüm kaynak örnek, bu şekilde, örnekleme hatasının azaltılması. Son olarak, bütün deneylerde kolayca kısa zaman dilimleri içinde yapılması gereken deney daha fazla sayıda izin veren, deney sonunda bir gün içinde tamamlanabilir. Ancak, hepsi hücre sayıları tedaviler, bazen özellikle hücresel ATP için bir araya geldi değil, bir varsayım sonra sinyal gücü orantılı kalması varsayımına dayanmasıdır. Hücreler artırmak veya tedavi sonrası boyutunda azalma Ayrıca, eğer, bu hücre sayısı etkilemeden sinyal gücünü etkileyebilir. Biz manuel sayıları dahil tüm canlılığı deneyleri, uyarılar bir dizi muzdarip olduğu sonucuna, ancak bilgisayarlı canlılığı deneyleri ilk yatırım değer. Her üç deneyleri kullanılarak birlikte hücresel yapısı ve fonksiyonu kapsamlı bir görünümünü verir.

Bu deneylerde oran sınırlayıcı bir faktör ATP deney süresi nispeten kısa olduğu için, kızılötesi boyama olduğunu. Kızılötesi tahlilleri için, sekiz 96-yuvalı plakalar (bkz. Şekil 1) dört plakaların her biri iki seri tarafından şaşırtıcı bir gün içinde, lekeli ve taranabilir tahmin ediyoruz. Bu tahmin, tespit bölgesinin 20 dakika varsayar, yıkama 30 dakika, yıkama 30 dakika, ardından 2 saat primer antikor inkübasyon bloke 30 dakika, yıkama 30 dakika süre ile 1 saat kul...

Watch this Scientific Journal Video about Viability Assays for Cells in Culture at JoVE.com

Viability Assays for Cells in Culture

Terapötik bileşikler genellikle ilk canlılığı deneyleri ile in vitro incelenmiştir. Bir insan gözlemci tarafından kör hücre sayımları hücre sayısındaki küçük değişikliklere son derece hassas olabilir ama fonksiyonunu değerlendirmek yok. Bilgisayarlı canlılığı deneyleri, burada açıklandığı gibi, objektif bir şekilde yapı ve fonksiyon hem de değerlendirebilir.

Kültür hücreleri için Canlılık Tahliller

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Research

JoVE Journal

Biology

46.0K Görüntüleme.  Duquesne University.  Terapötik bileşikler genellikle ilk canlılığı deneyleri ile in vitro incelenmiştir. Bir insan gözlemci tarafından kör hücre sayımları hücre sayısındaki küçük değişikliklere son derece hassas olabilir ama fonksiyonunu değerlendirmek yok. Bilgisayarlı canlılığı deneyleri, burada açıklandığı gibi, objektif bir şekilde yapı ve fonksiyon hem de değerlendirebilir. 

Video: Viability Assays for Cells in Culture

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Doku ve Hücre Sayım ve Canlılık Belirlenmesi

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Biyoloji

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

, Epilepsi Mutants Nöbetler Neurocircuit Tahliller Drosophila</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann-astrosit Etkileşimleri Analizi In Vitro Tahliller

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

Böbrek Hücreleri aşırı eksprese eden protein kinaz C izozimlerinin Mitokondriyal Fonksiyonlar ve Hücre Canlılık Değerlendirilmesi

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

İzlenmesi protein refolding için birleştiğinde Tahliller<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

Sendai-virüs ile İnsan Somatik Verimli Üretimi İnsan uyarılmış pluripotent kök hücrelerin

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

Kültürlenmiş memeli hücrelerinde büyük çapta paralel Raportör Tahliller

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Hücreler yanlış katlanmış proteinlerle degradasyonundan Tahliller

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...