우리는 ATP 분석에서 시약의 볼륨에 저장하는 아이디어 Juliann Jaumotte을 인정합니다. 우리는이 연구에 대한 재정 지원을 제공하는 메리 카루소, 뎁 WILLSON, 재키 파러의 약학 Mylan는 학교에 최상의 행정 지원에 대한 깊이 감사하고 있습니다. 감사는 또한 Hunkele 두려워한 질환 재단과 파킨슨 병 및 운동 장애 차 신경 세포 연구의 재정적 지원을위한 재단에 기인한다.

저자 중에 공개 할 충돌이 없습니다.

우리는 세 가지 생존 분석의 신호 강도가 선형 및 도금 밀도와 상관 관계가 있음을 발견했다. 그러나, 모든 분석은 2 배 또는 도금 밀도의 1.5 배의 변화에​​ 동등하게 구분합니다. N2A 세포의 경우, 적외선 분석은 특히 낮은 도금 밀도에서, ATP 분석보다 덜 민감하다. 적외선 분석이 ATP보다 덜 민감하지만, DRAQ5 + 사파이어 분석 및 α-튜 불린 분석은 그들이 N-아세틸 시스테인의 높은 보호에 미치는 ?...

생물 과학에서 가장 일반적인 생존 분석은 세포 수를 포함한다. 이것은 2013년 4월 29일 및 2013년 4월 30일의 키워드 중 하나 &quot;시험관&quot;또는 &quot;문화&quot;로 PubMed를 등장 최고 (가장 최근) (200) 출판물의 분석에 의해 입증된다. 이 간행물의 수동 세포 수의 계산을 포함하여 23.5 % 사용하는 세포 수의 분석은 자동화 된 세포 수는 이미징 소프트웨어를 사용하여 계산하고 트리 판 블루 배제. 라이브 / 데드 분석은이 책의 1 %에 사용되었다. MTT (3 - (4,5 - 디메틸 티아 졸 -2 - 일) -2,5 - 디 페닐 테트라 졸륨 브로마이드)하여 출판물의 개수 대사 생존력에 대한 분석은 11 %였다. 문학의이 조사는 또한 세포 수의 분석과 함께 같은 MTT 등의 분석을 사용하여 출판물의 수는 3.5 %였다 있음을 보여줍니다. 휴대폰 번호와 함께 세포 기능을 평가, 그 자체로 하나의 생존 분석을 사용하는 추세는 휴대 전을 평가하기위한 최선의 선택을 보인다에도 불구하고ntegrity. 나머지 세포가 기능 또는 그들이 잘 1,2에 존재에도 불구하고 건강한 수없는 경우가 있기 때문에 그 자체로 세포 수는 충분하지 않다. 반대로, 같은 ATP와 같은 기능적 수단 증가 또는 세포의 수가 병렬 변화의 부재에서 저하 될. 휴대폰 번호에서 대사 판독 값의 연결을 풀기는 ATP와 MTT 분석이 유일한 생존 분석으로 사용하지 말아야하는 것이 좋습니다. 본 보고서에서는, 세포 구조와 대사 기능을 모두 조사 세 생존 분석은 감당할 수있는 그 자체로 하나 분석보다 세포의 무결성을보다 포괄적 인 뷰에 대해 설명합니다. 우리의 분석의 두 개는 700과 800 nm의 채널에서 형광을 측정하는 적외선 이미 저를 필요로합니다. 잡음은 높은 신호 - 대 - 잡음 비 (3)에 이르는, 적외선 파장에서 낮다. 우리가 사용하는 오디세이 화상 카메라는 4.5 로그 동적 범위와 16 translatin의 비트 깊이적외선의 2 (16) 또는 65,536 그늘에 g. 이뿐만 아니라, 각각의 파장에 대해 색이 8 또는 256 음영을 수득 8 비트 컬러 이미징, 대조 할 수있다. 따라서, 16​​ 비트 영상은 미세한 해상도를 가지고 있습니다. 원래 적외선 이미지는 종종 프레젠테이션 게시 된 보고서에 (800 NM)과 적색 (700 nm의) 녹색 모조 착색되는 것을 주목해야한다. 오디세이 화상 카메라는 일반적으로 웨스턴 블로 팅과에서 셀 서부 4-7 모두에 사용됩니다. 에서 셀 포름 알데히드 고정 된 세포에 서부는 관심의 단백질에 대한 일차 항체를 사용하여 적외선 형광 보조 항체 차례로 레이블을 지정. 이 기술은 인산화 엔드 포인트 6에 특히 유용한 것으로 알려져있다. 우리의에서 셀 서부에서, 우리는 세포 골격 단백질 α-튜 불린 또는 800 nm의 채널에서 신경 미세 소관 관련 단백질 2 (MAP2)에 대한 고정 된 세포를 얼룩. 이들 단백질은 높은 신호 - 대 - 잡음 비를 수득 할 정도로 풍부하다. 우리는 또한 700에있는 우리의 판을 얼룩DRAQ5의 얼룩과 사파이어 얼룩 세포질에 대한 핵에 대한 나노 채널. 세포 골격 단백질과 DRAQ5 + 사파이어 얼룩 두 따라서 세포 구조를 반영합니다. 제 생존력 분석은 대사 기능을 측정하여 호출 &quot;셀 역가 글로.&quot;이 루시페라아제 기반 분석에서, 발광 값 ATP 농도에 정비례한다. ATP 분석은 일반적으로 가능한 세포에게 8-12을 정량화하는 데 사용됩니다. 셀 당 ATP 출력 독소 치료의 함수로 변경할 수 있기 때문에, 분석의 이름으로 단어 &quot;역가&quot;를 포함하는 것은 다소 명칭이 잘못된 것입니다 및 휴대폰 번호 8에 비례하므로 항상이다. ATP 농도는 13 시간주기 리듬에 의해 세포 분열 (14) 및 세포 분화 (15)에 의해 영향을받을 수있다. ATP 대사의 강력한 측정하기 때문에 그럼에도 불구하고, 여기에 표시된 ATP 분석을 수행 간단하고 유용합니다생존 16-21, 자체 수를 셀하지 않을 경우. 서부 따라서 혼자 하나 분석보다 세포의 무결성을보다 포괄적 인 사진을 얻을 수에서 셀 적외선을 보완하기 위해이 분석을 사용하여.

<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

현미경에서 수동 세포 수는 세포 생존 능력을 평가하는 중요한 수단이지만 시간이 많이 소요되므로 고가이다. 컴퓨터 생존 분석 장비의 측면에서 비용이 더 빠르고 더 객관적인 수동 세포 수보다 수 있습니다. 본 보고서는 이러한 세 생존 분석의 사용을 설명합니다. 이러한 분석의 두 적외선이며, 하나는 발광입니다. 두 적외선 분석은 16 비트 오디세이 이미 저에 의존하고 있습니다. 한 적외선 분석은 세포 기질에 대한 사파이어의 얼룩과 함께 핵의 DRAQ5 얼룩을 사용하고 700 nm의 채널을 시각화한다. 다른 적외선 분석, 인 - 셀 양식, 세포 골격 단백질 (α-tubulin의 또는 미세 소관 관련 단백질 2)에 대한 항체를 사용하여 800 nm의 채널을 레이블. 세 번째 생존 분석은 ATP를 위해 일반적으로 사용되는 발광 분석이지만, 우리는 비용을 절약하는 데 권장되는 볼륨의 내무반을 사용합니다. 이들 측정은 모두 선형 및 세륨의 개수와 상관 관계LLS 도금,하지만 감도에 따라 다릅니다. 세 가지 분석법은 시간 소모적 현미경을 회피하고 전체를 잘 샘플링함으로써 샘플링 에러를 감소시킨다. 마지막으로, 분석의 모든 쉽게 짧은 시간 프레임 내에서 수행되는 실험의 많은 수 있도록, 실험의 단부 중 하나 일 이내에 완료 될 수있다. 그러나, 그들은 모두 세포 수는 치료, 때때로, 특히 셀룰러 ATP에 대한 충족되지 않은 가정 후, 신호 강도에 비례하여 유지한다는 가정에 의존한다. 세포가 증가하거나 치료 후 감소 사이즈면 더욱이,이 세포 수에 영향을주지 않고 신호 강도에 영향을 미칠 수있다. 우리는 수동 계산을 포함한 모든 가능성의 분석은,주의 사항의 숫자에서 고통 결론하지만 컴퓨터 생존 분석은 초기 투자 가치가 있습니다. 세 가지 분석을 함께 사용하면 세포의 구조와 기능에 대한 포괄적 인 뷰를 생성합니다.

이들 실험에서 속도 제한 요인은 ATP 분석이 비교적 짧은 기간이기 때문에, 적외선 염색법이다. 적외선 분석을 위해, 우리는 여덟 96 - 웰 플레이트 (그림 1 참조) 4 판 각각의 압도적 인 두 개의 일괄 처리에 의해 하루 만에 염색 검사 할 수 있다는 것을 기대하고 있습니다. 이 추정은 고정의 20 분을 가정, 세탁 30 분, 세척의 3​​0 분 뒤에 2 시간 차 항체 배양을 차단 30 분, 액체의 30 분 뒤...

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

Viability Assays for Cells in Culture

치료 화합물은 종종 첫 번째 생존 분석과 시험 관내에서 검사합니다. 인간의 관찰자에 의해 블라인드 세포 수는 세포 수의 작은 변화에 매우 민감 할 수 있지만 기능을 평가하지 않습니다. 컴퓨터 생존 분석, 여기에 설명 된대로 객관적인 구조와 기능을 모두 평가할 수 있습니다.

문화의 세포에 대한 생존 분석 실험

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 조회수.  Duquesne University.  치료 화합물은 종종 첫 번째 생존 분석과 시험 관내에서 검사합니다. 인간의 관찰자에 의해 블라인드 세포 수는 세포 수의 작은 변화에 매우 민감 할 수 있지만 기능을 평가하지 않습니다. 컴퓨터 생존 분석, 여기에 설명 된대로 객관적인 구조와 기능을 모두 평가할 수 있습니다. 

비디오: 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.

카운팅 및 교양 세포의 생존을 결정

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

연구

생물학

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.

의 간질 발작의 돌연변이에 대한 Neurocircuit의 Assays 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.

사용 Schwann - astrocyte 상호 작용 분석 체외에서 Assays

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%.

쥐 간장 세포의 분리 및 차 문화

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.

신장 세포 Overexpressing 단백질 키나제 C Isozymes에 Mitochondrial 기능 및 세포 생존 능력의 평가

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.

의 모니터링 단백질 접힘에 대한 결합 분석 실험<em&gt; 사카 cerevisiae의</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.

센다이 바이러스와 인간의 체세포의 효율적인 생성 인간 유도 만능 줄기 세포

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.

배양 된 포유 동물 세포에서 대규모 병렬 리포터 분석 실험

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.

Zebrafish의에서 콩팥 세포 독소 미세 주입은 급성 신장 손상을 모델링하기

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.

세포의 잘못 폴딩 된 단백질의 분해에 대한 분석

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