저자는 fluc/mrfp/sr39tk의 리포터 유전자의 선물 Sanjiv Gambhir 감사드립니다. 이 작품은 캐나다의 줄기 세포 네트워크 (SCN), 그리고 제시의 여행 재단의 지원을받는되었다.

1. C2C12 Myoblasts의 유지 관리 및 전파 <ol><li> 판 C2C12 75cm 2 플라스크에 myoblasts와 10 %의 최종 농도에 태아 소 혈청 (FBS)와 보충 높은 포도당 Dulbecco의 수정 된 이글의 세럼 (HG-DMEM)에 세포를 유지하고 있습니다. 이 myoblastic 인구를 고갈되므로 세포는 언제든지 합류가하는 것을 허용하지 않습니다. ° C 사용하기 전에 물을 욕조에 37 항상 따뜻한 매체 :. 매체는 매일 참고 변경해야합니다. </li><li> myoblasts이 약 80 % 합류가되면 새로운 플라스크에 통과 세포. 문화 매체를 대기음. 트립신 억제 혈청을 포함 문화 매체의 모든 흔적을 제거하는 4-5 ML 행크스 균형 소금 솔루션 (HBSS)으로 세포를 씻으십시오. 간단히 2-3 ML 0.25 % 술병에서 자기편 myoblasts을 해리하는 (w / V) 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) 솔루션과 세포 레이어를 씻어. 5 분에 37 ° C에서 보육의 트립신과 장소 플라스크를 대기음. </li><li> 이 incuba 동안기는 HG-DMEM/10 % FBS 매체의 구 ML로 새 플라스크를 준비합니다. trypsinization 후, 중간에있는 모든 세포의 수집을 보장하기 위해 셀 및 피펫 4-5 시간에 완벽한 매체 10 ML를 추가합니다. 새로운 플라스크에 세포 현탁액 1 ML을 추가하고 37 5 % CO 2에 품다 ° C. </li></ol> 2. C2C12 세포 Transfection <ol><li> 일단 세포는 Invitrogen에서 제조업체의 지침에 따라, confluency 50 %를 transfect에 도달했습니다. 간단히, 4.5 ML OPTI-가상 매체와 90 μl Lipofectamine 2000 시약 조화를 이루고 있습니다. 또 다른 관에서 4.5 ML OPTI-가상으로 36 μg CMV-trifusion 기자 유전자 DNA가 조화를 이루고 있습니다. 5 분을 결합하고 기다리는 각 관을 긋기. 두 튜브의 내용을 결합하여, 부드럽게 혼합 정확히 삼십분 동안 실온에서 배양 참고 :. 두 번째 플라스크는 단지 부정적인 제어 역할을 Lipofectamine 및 OPTI-가상을받을 수 untransfected 셀에 설정해야합니다. </li><li> C2C12 세포에서 미디어를 제거하고 15.5을 추가신선한 HG-DMEM/10 % FBS 매체의 ML. 20 ML에 총 볼륨을 가져 transfection 매체를 추가 할 수 있습니다. </li><li> 세포가 37 최소한 20 시간을 위해 밤새 transfect 할 수 있도록 허용 ° C. </li><li> 다음 날, transfection 매체를 대기음 10 ML HG-DMEM/10 % FBS를 추가합니다. </li><li> 역 형광 현미경으로 세포를 볼 수 있습니다. 시야과 붉은 형광 이미지를 모두 캡처 (TRITC 필터 큐브를 이용하여, mrfp 예 / EM : 607분의 584 nm의). transfection 효율을 생성하는 뷰의 여러 분야에 대한 시야에서 볼 셀의 총 횟수로 나눈 형광에서 볼 RFP - 표현 세포의 수를 계산합니다. </li></ol> 3. 셀 Survivability / MTT 분석의 평가 <ol><li> 24 잘 플레이트의 각도에 transfection, 판 1x10 5 C2C12 세포하기 전에 어느 날. 셀은 HG-DMEM/10 % FBS 500 μl 권 도금해야합니다. </li><li> 다음 날, transfect의 myoblasts 하룻밤 이전에 설명 된대로. transfection 시약에 대한 제조업체의 제안에 따라24 잘 접시 t 권. 컨트롤에 불과 Lipofectamine (NO DNA)와 우물 세트를 품다. 그 적절한 transfection가 발생했습니다 보장하기 위해 형광에 따라 세포를 볼 수 있습니다. </li><li> HG-DMEM/10 % FBS 5 MG / ML thiazolyl 블루 tetrazolium 브로마이드 (MTT)에 transfection 매체 및 품다 myoblasts을 제거합니다. transfected 우물의 8 D-luciferin을 추가, 4 시간 동안 37 ° C에서 알을 품다. </li><li> 우물에서 매체를 제거합니다. 각도에 이소프로판올 180 μl를 추가하여 파란색 formazan의 결정을 Solubilize. 15 분에 37 ° C에서 흔들어. </li><li> 96 - 웰 플레이트에있는 침전물, 피펫 솔루션을 피하고 575 nm의에서 흡광도를 참조하십시오. </li></ol> 4. 이식을위한 Myoblasts의 작성 <ol><li> , 미디어를 제거 HBSS와 myoblasts을 씻고, 및 1.1에 명시된 바와 같이 셀을 trypsinize. </li><li> 전체 매체 중 4 ML의 세포를 다시 일시 중지합니다. </li><li> hemocytometer 사용 10 6 myoblasts를 포함하는 볼륨을 생성하기 위해 셀을 계산합니다. 피펫 권멸균 1.5 ML의 microtubes에 매화. ~ 10 %의 transfection 효율이 값은 100,000 루시 페라 제 - 표현 세포가 이식 후 GE ExploreOptix 스캐너에서 감지되고있는 것으로 나타났습니다. </li><li> 10 6 C2C12 세포를 포함, 15 μl의 최종 주입 볼륨을 충족해야합니다. 필요한 경우, 1 분에 2,000 rpm으로 microtubes을 원심 분리기. 피펫으로 표면에 뜨는을 자세히 대기음. HG-DMEM는 FBS를 부족의 15 μl로 세포를 다시 일시 중지합니다. </li></ol> 5. 세포 주입 <ol><li> 2퍼센트 2 / isoflurane % O 2 마우스를 마취. 등쪽의 뒷다리 영역에서 머리카락을 따. 1.5 % 2 / isoflurane % O 2 마취를 유지합니다. </li><li> 확인 C2C12 myoblasts도 일시 중지됩니다. 쉬운 위치에 마우스로, 뒷다리 다리를 확장하고 직접 30 ° 각도에서 gastrocnemius 근육의 외측 머리에 세포를 주입하는 인슐린 주사기를 사용합니다. 오른쪽 뒷다리와 untransfected myoblasts의 정수로 transfected myoblasts를 삽입contralateral (왼쪽) 뒷다리 다리를 O. </li><li> 기준 bioluminescence 검사를 수행 빠르게 발생하기 쉬운 위치에 마우스를 놓고, 광학 스캐너에 무대에 마우스를 전송할 수 있습니다. 마취 줄을 연결. 마우스가 anesthetized 상태로 유지하려면, 두 사람이 마취 라인을 연결하기 위해 존재해야합니다 스캐너의 다른 장소 동물 동안. </li><li> 분사의 영역이 표시되도록 부드럽게 뒷다리 다리를 확장합니다. 예를 들어, 의료 테이프와 같은 부드러운 접착제와 장소에 테이프를 두 다리. </li><li> 챔버를 닫고이 감지 배경 신호를 증가로 더 빛이, 스캐너의 내부에 액세스 할 수 없습니다 있는지 확인합니다. 스캐너는 bioluminescence 이미징에 대한 제조업체의 지침에 포함 된 매개 변수를 설정해야합니다. 특히, &quot;더 이상 레이저&quot;를 선택하지 않습니다 보장합니다. </li><li> 뽑혀 지역 및 시작 검사 주변 관심 (ROI)의 영역을 그립니다. </li></ol> 6. MDX 마우스로 Fluc 기판, D-luciferin의 주입 &lt;좋았어요&gt; <li> 마우스가 anesthetized intraperitoneally는 반딧불 루시 페라 제 기판, D-luciferin의 150 밀리그램 / kg를 주입하는 동안 (40 MG / ML 주식 솔루션에서, 제조업체의 지침에 따라 구성). </li><li> 마우스를 복구하고 다음 스캔을 위해 마우스를 준비하기 전에 15 분 정도 호응 기간을 허용합니다. </li></ol> 7. BLI는 DMD의 마우스 모델에 임플란트 후 루크 - 표현 MPCs를 타겟팅 할 수 <ol><li> 5.1에서 설명 된대로 이해 기간 후, 다시 마우스를 마취. </li><li> 광학 스캐너에 다시 마우스를 전송하고 백그라운드 검색과 같은 방식으로 다른 bioluminescence 검색을 수행합니다. </li><li> 20 분 통풍 관 기간이 최대 신호 강도를 제공해야하지만, 이후의 스캔이 수행 될 수 있습니다. </li><li> 이미지 수집이 완료되면, 당신의 기관 동물 윤리위원회 및 동물 관리 (CCAC)의 캐나다 협의회가 설정 한 지침에 따라 마우스를 희생. 내가 바로 뒷다리의 근육과 장소를 분리N 포르말린 10 % 파라핀 근입을 수정합니다. myoblasts의 근육 주사를 확인하는 루시 페라 제에 대한 immunohistochemistry 착색을 수행합니다. </li></ol>

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저자는 공개 할 수 없어요.

본 연구에서는, 우리는 이식 후 비 invasively 대상 myoblasts / MPCs에 신속하고 신뢰할 수있는 분자 영상, 리포터 유전자의 접근 방식을 설명하고 있습니다. 이 연구는 bioluminescense 이미징 (BLI), 사실은, 쉽게 세포의 주입을 통해 세포 engraftment의 길이 방향 평가에 적용 할 수있는 세포를 타겟으로하는 방식을 통해 이식 MPCs의 단기 현지화을 보여줍니다 동안 안정적으로 명시 리포터 유전자. ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> C2C12 myoblasts </td><td> ATCC </td><td></td><td></td></tr><tr><td> Dulbecco의 수정 된 이글의 매체 </td><td> 생명 기술 </td><td> 12800-017 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> 생명 기술 </td><td> 12483020 </td><td></td></tr><tr><td> 0.25 % 트립신 - EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 생명 기술 </td><td> 25200-72 </td><td></td></tr><tr><td> 행크스 균형 소금 솔루션 </td><td> 시그마 알드리치 </td><td> H6648 </td><td></td></tr><tr><td> Lipofectamine 2000 </td><td> 생명 기술 </td><td> 11668-019 </td><td></td></tr><tr><td> 니콘 이클립스 TE2000-5 </td><td> 니콘 인스트루먼트 주식회사 </td><td></td><td></td></tr><tr><td> Xenolight D-luciferin </td><td> PerkinElmer </td><td> 122796 </td><td> PBS 40 MG / ML </td></tr></tbody></table>

molecular imaging to target transplanted muscle progenitor cells

Duchenne 근육질 영양 장애 (DMD)는 3500 소년의 1에 영향을 미치는 심각한 유전 신경 근육 장애이며, 진보적 인 근육 퇴행 1, 2에 의해 특징입니다. 환자에서 손상된 myofibers를 다시 생성 할 수 주민 근육 위성 세포의 능력 (SCS)는 4 점점 비효율적이된다. 따라서, 건강 분야에서 근육 전구 세포 (MPCs) / myoblasts의 이식은 DMD에 유망한 치료 방법이다. 줄기 세포 치료의 사용에 대한 주요 제한은, 그러나, 이식 세포의 장기 모니터링을위한, 그 효과를 평가하기위한 신뢰할 수있는 이미징 기술의 부족이다. 여기, 우리는 myoblast 이식의 성공 여부를 평가하는 비 침습적, 실시간 접근 방식을 설명합니다. 이 방법은 (반딧불 루시 페라 [fluc], monomeric 붉은 색 형광 단백질 [mrfp]와 sr39 thymidine의 키나제 [sr39tk]) expre 유전자로 구성된 통합 된 퓨전 리포터 유전자 활용ssion은 여러 이미징 modalities 9, 10으로 이미징 할 수 있습니다. 양전자 방출 단층 촬영 (PET), 단일 광자 방출 계산 된 단층 촬영 (SPECT), 자기 공명 영상 (MRI), 광학 이미징, 높은 주파수 3D-초음파 등의 영상 modalities의 다양한 고유의 장점과 한계를 각각, 지금 사용할 수 있습니다 11. Bioluminescence 이미징 (BLI) 연구 예를 들어, 상대적으로 낮은 비용과 높은 처리량가되는 장점이 있습니다. 그것은이 연구에서, 우리는 마우스에 주입 후 가능한 C2C12 myoblasts의 단기 현지화를위한 융합 유전자 및 bioluminescence 이미징 (BLI) 내에 포함 된 반딧불 루시 페라 제 (fluc) 리포터 유전자 시퀀스를 이용, 이런 이유로 DMD의 모델 12-14 (X 염색체 [MDX] 마우스에 근육 영양 장애). 중요한 것은, BLI라는 MPCs 후 주입의 동력학을 검토 할 수있는 수단으로 우리를 제공하며, t를 통해 반복적으로 세포를 추적 할 도움이 될 것입니다IME와 다음과 같은 마이그레이션. 우리 리포터 유전자 접근 방식이 더 우리는 하나의 생활 주제에 여러 이미징 modalities를 병합 할 수 있습니다, 단층 자연, 훌륭한 공간 해상도와 큰 동물과 인간 10,11까지 확장 할 수있는 능력에 따라, PET 미래 작업의 기초를 형성 할 우리 제안은 잠복기 모델 및 임상 응용 프로그램에 세포 개발 방법의 급속한 번역을 촉진 할 수 있습니다.

50~60% confluency시, C2C12 myoblasts는 transiently 위에서 언급 한 퓨전 기자 유전자가 반딧불 루시 페라로 구성된 구조와 transfected되었습니다 [fluc], monomeric 붉은 색 형광 단백질 [mrfp]와 sr39 thymidine 키나제 [sr39tk] (그림 1A). Transfection 효율은 건설 기자의 mrfp 시퀀스의 활용, 형광 현미경 (인물 1B, C)을 통해 계산되었다. 셀 survivability은 BLI 기판, D-luciferin <strong...

Watch this Scientific Journal Video about Molecular Imaging to Target Transplanted Muscle Progenitor Cells at JoVE.com

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

myoblast 이식의 성공 여부를 평가하는 비 침습적 방법이 설명되어 있습니다. 방법은 그의 표현 다른 이미징 modalities를 이미징 할 수 있습니다 유전자로 구성된 통합 된 퓨전 리포터 유전자의 장점을 걸립니다. 여기, 우리는 사용을<em&gt; fluc</embioluminescence 이미징을 통해 세포를 타겟팅 할 수&gt; 리포터 유전자 시퀀스.

이식 근육 전구 세포를 타겟팅 할 수 분자 이미징

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

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9.1K 조회수.  Lawson Health Research Institute. myoblast 이식의 성공 여부를 평가하는 비 침습적 방법이 설명되어 있습니다. 방법은 그의 표현 다른 이미징 modalities를 이미징 할 수 있습니다 유전자로 구성된 통합 된 퓨전 리포터 유전자의 장점을 걸립니다. 여기, 우리는 사용을 fluc 리포터 유전자 시퀀스.

비디오: Molecular Imaging to Target Transplanted Muscle Progenitor Cells

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

염증성 플라크의 적외선 형광 (NIRF) Intravascular 분자 이미징, Multimodal 접근 가까운 생체내에서 죽상 경화증의 영상을

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

연구

의학

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Ferumoxytol, FDA 승인 철 산화물 Nanoparticle과 함께 줄기 세포를 라벨링

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

The Use of Thermal Infra-Red Imaging to Detect Delayed Onset Muscle Soreness

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically inactive for prolonged periods of time, and begin with an unexpected bout of exercise1-4, but can also occur in athletes who exercise beyond their normal limits of training5. The symptoms associated with this painful phenomenon can range from slight muscle tenderness, to severe debilitating pain1,3,5. The intensity of these symptoms and the related discomfort increases within the first 24 hours following the termination of the exercise, and peaks between 24 to 72 hours post exercise1,3. For this reason, DOMS is one of the most common recurrent forms of sports injury that can affect an individual&#x2019;s performance, and become intimidating for many1,4.
For the last 3 decades, the DOMS phenomenon has gained a considerable amount of interest amongst researchers and specialists in exercise physiology, sports, and rehabilitation fields6. There has been a variety of published studies investigating this painful occurrence in regards to its underlying mechanisms, treatment interventions, and preventive strategies1-5,7-12. However, it is evident from the literature that DOMS is not an easy pathology to quantify, as there is a wide amount of variability between the measurement tools and methods used to quantify this condition6. It is obvious that no agreement has been made on one best evaluation measure for DOMS, which makes it difficult to verify whether a specific intervention really helps in decreasing the symptoms associated with this type of soreness or not. Thus, DOMS can be seen as somewhat ambiguous, because many studies depend on measuring soreness using a visual analog scale (VAS)10,13-15, which is a subjective rather than an objective measure. Even though needle biopsies of the muscle, and blood levels of myofibre proteins might be considered a gold standard to some6, large variations in some of these blood proteins have been documented 6,16, in addition to the high risks sometimes associated with invasive techniques. 
Therefore, in the current investigation, we tested a thermal infra-red (IR) imaging technique of the skin above the exercised muscle to detect the associated muscle soreness. Infra-red thermography has been used, and found to be successful in detecting different types of diseases and infections since the 1950&#x2019;s17. But surprisingly, near to nothing has been done on DOMS and changes in skin temperature. The main purpose of this investigation was to examine changes in DOMS using this safe and non-invasive technique.

The purpose of this investigation was to assess whether using an infra-red thermal camera is a valid tool for detecting and quantifying the muscle soreness after exercising.

지연 발병 근육 쓰림을 감지하는 열 적외선 이미징의 사용

Delayed onset muscle soreness (DOMS), also known as exercise induced muscle damage (EIMD), is commonly experienced in individuals who have been physically ...

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.

To demonstrate MR cancer molecular imaging with a small peptide targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma in a mouse prostate cancer model.

작은 분자 CLT1 펩타이드 표적 조영제와 전립선 암의 MR 분자 영상

Tumor extracellular  matrix has abundance of cancer related proteins that can be used as biomarkers  for cancer molecular imaging. In this work,  we ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

인간 혈청에있는 N-메틸-D-아스파 라진 산염 수용체 또는 도파민 2 수용체에 대한 항체를 검출하는 높은 처리량 유동 세포 계측법 세포 기반 분석

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

순차<em&gt; 생체 내</em&gt; 골절 치료시 골 형성 줄기 / 전구 세포의 이미징

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

골격근 질환의 정량적 자기 공명 영상

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

대동맥 석회화와 염증의 혈관 평활근 세포 및 이미지의 석회화

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

Contractility Measurements of Human Uterine Smooth Muscle to Aid Drug Development

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We describe methods to measure ex vivo myometrium contractility. This assay can be used to investigate factors and molecules involved in the modulation of myometrial contraction and to determine their excitatory or inhibitory actions, and hence their therapeutic potential in vivo. Biopsies are obtained from women undergoing cesarean section delivery with informed consent. Fine strips of myometrium are dissected, clipped and attached to a force transducer within 1 mL organ baths superfused with physiological saline solution at 37 &#176;C. Strips develop spontaneous contractions within 2&#8211;3 h under set tension and remain stable for many hours (&#62;6 h). Strips can also be stimulated to contract such as by the endogenous hormones, oxytocin and vasopressin, which cause concentration-dependent modulation of contraction frequency, force and duration, to more closely resemble contractions in labor. Hence, the effect of known and novel drug leads can be tested on spontaneous and agonist-induced contractions.
This protocol specifically details how this assay can be used to determine the potency of known and novel agents by measuring their effects on various parameters of human myometrial contraction. We use the oxytocin- and V1a receptor antagonists, atosiban and SR49059 as examples of known compounds which inhibit oxytocin- and vasopressin-induced contractions, and demonstrate how this method can be used to complement and validate pharmacological data obtained from cell-based assays to aid drug development. The effects of novel agonists in comparison to oxytocin and vasopressin can also be characterized. Whilst we use the example of the oxytocin/ vasopressin system, this method can also be used to study other receptors and ion channels that play a role in uterine contraction and relaxation to advance the understanding of human uterine physiology and pathophysiology.

This article describes experimental protocols to study ex vivo contractions of human myometrium and their application in drug discovery. This technique is used to improve the understanding of myometrial physiology and pathophysiology as well as to validate pharmacological data from novel research probes or drug leads.

보조 약물 개발에 인간 자 궁 평활 근의 수축 측정

Discovery and characterization of novel pharmaceutical compounds or biochemical probes rely on robust and physiologically relevant assay systems. We ...