K. 웡, STC 왕은 기능 및 분자 이미징 센터, 브리검 앤드 위민스 병원에서 기금에서 지원하는 K. 웡, STC 왕은 생명 공학 및 정보학, 메소 디스트 병원 연구소, 웨일 코넬 메디컬 칼리지를위한 센터에 의해 지원됩니다. KK 크왕, 난 첸, JQ 르네는 바이오 메디컬 이미징, 매사 추세츠 종합 병원에 대한 Athinoula A. Martinos 센터에서 기금에 의해 지원됩니다. Lenuta Kloetzer, 브래든 쿠오는 Neuroenteric 연구 센터, 매사 추세츠 종합 병원의 기금에 의해 지원됩니다 저자 DRS 감사합니다. Q. Zeng 및 광학 이미징 연구소, 브리검 및 기술 지원을위한 여성 병원의 X. 쑤.

<ol><li> 여성 HO - 1 - 루시페라제 유전자 변형 생쥐 (4-6 주 세)가 도착되면, 주식 타코닉 팜에서 구입할 수 있으며, 생쥐는 숙박 시설을 허용하는 최소 4 일 동안 동물의 주택 시설에 배치됩니다. </li><li> bioluminescence 이미징을위한 준비 : <ol style="list-style-type:lower-alpha;"><li> 각 HO - 1 마우스의 무게를. 동물의 무게 luciferin의 복용량을 계산이 필요합니다. </li><li> 제모 : 또는 다시 동물의 복부 및 /여 머리 제거제 나이르 ®를 적용하는 면으로 스왑을 사용합니다. 5-10초 기다 리면서. 머리를 닦아으로 깨끗한 면봉 스왑을 사용합니다. 깨끗한 남은 머리를 닦아 증류수에 조직의 한 조각을 찍어. </li><li> 배경 잡음을 줄이기 위해 IVIS 100 역의 이미징 플랫폼 이외의 형광 검은 종이 (스트라 스 모어 Artagain ® 검은 종이)를 놓으십시오. </li><li> luciferin (Xenogen 회사에서) 7.5 MG / ML (멸균 H 2 O에 용해)의 농도에 솔루션을 준비합니다. luciferin의 복용은 65.5 밀리그램 / kg 체중이다. 따라서, 20g의 마우스 0.175 ML luciferin 솔루션의 intraperitoneal 주사를 필요로합니다. 이것을 달성하기 위해, luciferin 솔루션 (0.175ml)의 단일 사출 량은 26 게이지 바늘로 주사기에 미리입니다 </li></ol></li><li> 규아 샤 절차 : 규아 샤는 bioluminescence 이미징 프로토콜을 실행하기 전에 한 번만 적용됩니다. 규아 샤는 통증이 아니므로, 마우스는 진정 isoflurane으로 간단히 anesthetized해야합니다. 규아 샤 절차는 다음 단계를 포함 : <ol style="list-style-type:lower-alpha;"><li> 규아 샤 절차 중 규아 샤에 의해 몇 시간을 목표로 피부 부분에 기름칠을하는 요리 기름이나 증류수를 적용합니다. </li><li> 반복 세라믹 수프 스푼이나 플라스틱 숟가락을 사용하여 부드러운하지만 회사 강제에서 마우스 뒤쪽의 머리카락이없는 지역을 다쳤어요. </li><li> 다시 피부가 빨간색 때까지​​ 힘든 계속, 어떤은 일반적으로 2-3분 이내에 달성 피하 혈액 넘쳐 흐름의 표시입니다. </li></ol></li><li> HO - 1 upregulation은 몇 시간에 걸쳐 천천히 쌓아이므로 Bioluminescence 이미징은 규아 샤 후 즉시 또는 몇 시간이 시작될 수 있습니다. bioluminescence 이미징을위한 절차는 다음과 같습니다 <ol style="list-style-type:lower-alpha;"><li> isoflurane (1.5 %) 및 ​​의료 등급 공기의 혼합물로 채워진 마취 유도 챔버에 마우스를 마취. </li><li> 마우스 anesthetized되면​​, IVIS 100 광학 이미징 역에서 이미징 챔버 마우스를 이동합니다. 나태한 자세 (최대 복부)에 마우스를 위치. 이미징 챔버가 계속 isoflurane의 1.5 %로 들어갈 수 있습니다. 이미징 플랫폼은 37 가열 ° C에서 마우스 보온 수 있습니다. </li><li> &quot;중간 비닝 &#39;에서 이미지 수집 30 초 설정 노출 시간을 설정합니다. 이미지를 얻기 시작합니다. 수동 또는 자동으로 이미지 획득 매일 3 분 반복하는 기계를 설정합니다. 첫 번째 이미지의 취득 후, 관심 (ROI)의 지역은 복부, 가슴 및 머리 영역을 커버하기 위해 그려진 것입니다. 이것은 투자 수익 (ROI)은 다음 복사와 &quot;생활 이미지 ®&quot;IVIS 100 스테이션을 함께 소프트웨어를 사용하여 다음과 같은 이미지에 붙여넣을 수 있습니다. </li><li> 신호 강도는 초당 광자 단위로 측정됩니다. 마크 시간은 신호 강도에 대한 최대 가치를 도달하고 최고 시간 이후 약 5-10분위한 이미징 마우스 계속. </li><li> 부정사 위치에 이미지 인수가 완료되면, 마우스를 뒤집과 발생하기 쉬운 위치 (백업)의 그것을 믿는다. 이제 소프트웨어 &quot;생활 이미지 ®&quot;의 사용에 다시하고 머리 영역을 커버하기 위해 그린 투자 수익 (ROI)과 5-10분위한 이미징 동물을 계속합니다. 이미지의 선택은 나태한 자세 후 쓰러진 자세는 임의입니다. 하나는 관심의 주요 장기에 따라 발생하기 쉬운 위치로 시작하도록 선택할 수 있습니다. 또 다른 옵션은 이미지를 별도의 실험에서 부정사와 발생하기 쉬운 위치를하는 것입니다. </li><li> 쓰러진 자세에서 이미징 인수가 완료되면, isoflurane 끄고 복구 유도 실로 이미징 챔버에서 마우스를 이동합니다. 유도 챔버는 현재 의료에만 공기와 마우스는 보통 미만 분 깨어나서 함께 들어갈 수 있습니다. </li><li> 후처리에 대한 영상 자료를 저장합니다. </li><li> 초기 이미징 연구 이후, 추가 영상 인수는 규아 샤 후 몇 일 동안 실시됩니다. 하나의 샘플 일정은 12 일 시간, 24 번째 시간, 36 번째 시간, 48 번째 시간, 72 차 시간 120 일 시간에 영상을 반복하지만 이미징 일정의 정확한 타이밍은 유연성입니다. 더 이상 후속이 필요한 경우 추가 이미지 세션 추가할 수 있습니다. 이미징 전에 머리를 다시 성장이있다면 절차를 제거하는 머리를 반복합니다. </li></ol></li><li> 컨트롤 그룹에 대한 모든 절차를 반복하지만 규아 샤 (3 단계)를 건너 뛰십시오. 생쥐 같은 그룹은 제어와 규아 샤, 제어 EXP 모두 사용하는 경우eriment는 한 달쯤 후에 규아 샤 절차 전에 선호 수행하거나해야합니다. </li></ol> 대표 결과 : 규아 샤에 대응 HO - 1의 생체내 upregulation 그림의 bioluminescence 이미지 1 보여줍니다. 그림 2의 그래프는 규아 샤와 관련된 동일한 마우스의 몸 전체에서 광학 흐름 (광자 / 초) 120 시간 이상의 양적 시간적 변화를 보여주는 ... <img src="/files/ftp_upload/1385/figure1tmb.jpg" alt="그림 1" border="0" /> 그림 1. 왼쪽부터 각각 18시간 36 시간 1백20시간 게시 규아 샤에서 규아 샤 전에 같은 마우스의 전면보기 (부정사)의 권리, 대표 이미지, 있습니다. 규아 샤 후, 하나는 위장관, 성기 트랙트, 간, 신장 (뒷면보기에서 표시되지 않습니다), 그리고 다른 사람의 영역을 포괄 여러 장기에 상당한 신호 강도 증가의 진행 상황을 관찰. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1385/figure1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 위해. <img src="/files/ftp_upload/1385/figure2tmb.jpg" alt="그림 2" border="0" /> 그림 2. 그림 1 같은 마우스 규아 샤에 따라 120시간 이상 추적 몸 전체에서 자속의 양적 변화 (광자 / 초). 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1385/figure2.jpg">여기를 클릭</a> 그림 2의 더 큰 버전을 볼 수 있습니다.

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The authors have nothing to disclose.

HO - 1, 헴 oxygenase의 inducible 양식의 전사는 헴, 과산화수소, UV 조사, hypoxia, 물리적 스트레스 등 많은 요인에 의해 upregulated 있습니다. 이러한 보고된 HO - 1 bioluminescence 프로토콜로 온몸의 화상은 여러 장기에서 체계적 HO - 1 표현의 빠른 스냅샷을 제공합니다. 작은 동물에서, bioluminescence 이미징은 높은 감도에 - 생체내 유전자 발현 체계의 변경을 실시간으로 양적 세로 광학 평가, 그림과 같이 있습니?...

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<td>Luciferin</td>
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<td>IVIS 100</td>
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<td>Caliper </td>
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<td>Previously Xenogen</td>
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bioluminescence imaging of heme oxygenase-1 upregulation in the gua sha procedure

규아 샤는 피하 microvascular 혈액 넘쳐 흐름과 타박상을 일으킬 힘든 피부를 고용하고 중국의 전통 민속 요법입니다. 의 발광 생물 광학 이미징을위한 프로토콜 HO - 1 -<em&gt; 루시페라제</em이 논문에 보고된&gt; 유전자 변형 생쥐는 급속한를 제공합니다<em&gt; 생체내에</em의 upregulation의&gt; 분석 헴 oxygenase - 1 (HO - 1) 규아 샤 절차에 대한 응답으로 유전자 발현. HO - 1 길이 산화 스트레스에 대한 cytoprotection를 제공하는 것으로 알려져있다. bioluminescence 출력에 의해 평가 HO - 1의 upregulation은, 규아 샤 발표 순환 헤모글로빈 제품 산화 반응을 나타내는 것으로 생각됩니다. 규아 샤는 점상 (스플린터 출혈) 또는 피하 모세 혈관에서 혈액의 넘쳐 흐름을 나타내는 출혈 (멍) 관찰 때까지 뒤로 또는 유전자 변형 마우스의 다른 목표 부위에 윤활 피부를 통해 부드러운 스푼의 가장자리 반복 스트로크에 의해 관리되었다. 규아 샤 후, bioluminescence 이미징 세션은 여러 내부 장기에 HO - 1 표현의 역학에 따라 몇 일 동안 매일 실시했다.

Watch this Scientific Journal Video about Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure at JoVE.com

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

규아 샤, 중국어 번체 치료 피부 스크레이핑은 피하 microvascular 혈액 넘쳐 흐름을 발생합니다. 우리의 bioluminescence 이미징의 프로토콜보고 HO - 1 -<em&gt; 루시페라제</em규아 샤는 헴을 upregulates는 것을 입증하는&gt; 유전자 변형 생쥐 oxygenase - 1 여러 장기 (HO - 1).

규아 샤 프로 시저에 헴 Oxygenase - 1 Upregulation의 Bioluminescence 이미징

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

bioluminescence-imaging-heme-oxygenase-1-upregulation-gua-sha

Research

JoVE Journal

Biology

13.9K 조회수.  Massachusetts General Hospital, Harvard Medical School. 규아 샤, 중국어 번체 치료 피부 스크레이핑은 피하 microvascular 혈액 넘쳐 흐름을 발생합니다. 우리의 bioluminescence 이미징의 프로토콜보고 HO - 1 - 루시페라제 유전자 변형 생쥐 oxygenase - 1 여러 장기 (HO - 1).

비디오: Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

만성 뇌 영상에 대한 Craniotomy 외과 절차

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

연구

생물학

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

체외에서 인간 배아 줄기 세포의 생체내의 Bioluminescence 리포터 진 영상에

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Murine 신장 이식 절차

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

FDG micro-PET/CT와 마우스의 브라운 지방의 기능 영상

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

루시 페라 제 Bioluminescence 기자를 사용하여 유전자 발현의 셀 자치 Circadian 시계 리듬을 모니터링

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

사멸 세포 간극의 이미지를하는 동안 라이브<em&gt; 초파리</em&gt; 배아

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

포유 동물 세포에서 헴 합성 레벨 측정

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

고해상도 시간 경과 이미징 및 생활 인간 제대 정맥 내피 세포의 미세 소관 역학의 자동 분석

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

mtHyper7을 사용한 살아있는 효모 세포의 미토콘드리아 과산화물 이미징

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

저자 스포트라이트: 미토콘드리아 연구의 발전 - 세포 내 분석을 위한 mtHyper7 바이오센서 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

살아있는 효모 세포에서 미토콘드리아 과산화물을 위한 비율계량 바이오센서인 mtHyPer7의 이미징

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...