건강, NHLBI 국립 연구소 (R &amp; D, PY, PS). 미국 심장 협회 (JL). 스탠포드 대학 VPUE 그랜트 (JL).

이전 연구 1에서 적응. 1. SPIO에 Annexin V의 활용 <ol><li> 2 : 0.15 M 나트륨 periodate (NaIO 4) (중량 비율 4시 1분 무게)와 솔루션 어둠 속에서 20 1 시간을위한 SPIO 입자 (오션 나노기술 주식 회사, 5 밀리그램 / ML) ° C를 산화. </li><li> 20 ° C.에 0.15 M 나트륨 borate (나 2 B 4 O 7 10H 2 O)에 : ANX 단백질을 (체중 1시 1분 비율, 체중) 정화와 함께 12 시간을위한 산화 SPIO를 품어 </li><li> 연기 후드 나트륨 borohydride (NaBH 4)와 1 시간에 대한 혼합물을 절감하고 0.15 M 트리스-HCL과 끄다. </li><li> 14,000 XG (75 μm의 기공 Microcon 필터, Millipore, Billerica, MA)에서 원심 분리하여 ANX-SPIO에서 무료 ANX를 분리합니다. </li><li> 또한 마그네틱 구분 장치 (오션 나노기술 주식 회사)를 사용하여 언바운드 불순물을 제거하여 화합물을 수정하십시오. </li><li> 필터 retentate (ANX-SPIO) 단백질 수준을 수량-80에서 브래드 포드 단백질 분석 및 점포별로 ° C에서 2 시에 1% 혈청 알부민과 프로 테아제 억제제 (정지 프로 테아제 억제제, 열 과학, 1-50 밀리그램 / MG ANX)와 0.1 M 트리스-HCL의 MG / ML. </li></ol> 2. 동적 라이트 산란 <ol><li> 별도로 0.1의 광학 밀도에 PBS에서 SPIO와 ANX-SPIO를 희석하고 Zetasizer 나노 DL을 기계 (Malvern 주식, Worcestershire, 영국) 3에서 동적 광 산란에 의해 분석합니다. </li><li> Z-평균 입자 크기와 polydispersity 지수 (PDI)로, 화합물을위한 monomodal 봉우리 (이상적으로) 구합니다. 혼자 철 산화물 nanoparticle의 DL을 - 측정된 크기로이 크기를 비교합니다. ANX-SPIO DL을 결과 동질 ANX-SPIO 수종 3 반영, 0.13 1 PDI와 78 nm의의 켤레축 입자 유체 직경을 보여주었다. </li></ol> 3. 세포 배양, 문화 Apoptosis의 유도 및 교양 세포의 MRI <ol><li> 문화 성인용 마우스 골수 mesenchymal의DMEM에서 편 세포 (mMSCs가) 10 % FBS와 선호 통과에 1퍼센트 페니실린 - 스트렙토 마이신 (Invitrogen 주식 회사), 4월 20일에서 30일까지으로 보충. </li><li> DOX (시그마, 0.9 %의 식염수에 1 ㎜, 미디어 1 μm의 농도) 12와 37 ° C에서 시간 변화에 대한 치료로 문화 세포의 apoptosis를 유도. </li><li> ANX-SPIO (아래 3.4에서 설명)와 MRI 또는​​ TUNEL 얼룩 후 현미경을 사용하여 (APO-BrdUTM TUNEL 분석 키트, 분자 프로브 / Invitrogen, 유진, OR) 또는 ANX-FITC와 얼룩 후 중 하나를 사용하는 문화에 apoptosis의 양을 계량 (로슈, 스위스). </li><li> 37 15 분 ° C. 들어, ANX-SPIO (명시된 바와 제외한 10 MG 단백질 / ML 매체)로 또는 무료 SPIO (5 밀리그램)의 동등한 무게와 세포를 분류 SPIO의 nanoparticles의 확산을 막기 위해 1퍼센트 아가로 오스 1 ML 4 개의 PBS와 시간, 오버레이를 씻으십시오. 요리의 MRI (아래 설명) 수행합니다. 또는 세포를 씻고 DOX 및 ANX-SPIO 라벨 이후 trypsinize 다음, 전지가 허용Eppendorf 튜브에 알약을 형성하고 있습니다. </li></ol> 4. 체외 MRI에서 <ol><li> 체외 MRI 분석에 대한 표준 무릎 수신기 코일과 1.5 ~ 3 테슬라 GE Signa의 자극 스캐너를 사용하십시오. </li><li> 반복 시간 (TR) 550 MS, 에코 시간 (TE) 10 MS, 플립 각도 15 °, 매트릭스 256 X 256, 현장 : 문화 요리 또는 튜브 한천 팬텀으로, 그리고 그라디언트 - 에코 철부지 그라디언트 시퀀스를 사용하여 이미지를 놓으십시오 의 뷰 3cm X 3cm, 슬라이스 두께 1.3 mm, 평균 횟수 (NEX) 2, 간격 0mm 5. 이미지 axially 고정 영역의 지역 -의 이익을 사용하여 T2 * 신호 손실 (ROIs) (예를 들어, 0.8 mm 2) 접시 세포 계층을 통해. 한천 팬텀에서 투자 수익 (ROI) 제어 신호를 사용하여 배경에 조정합니다. 유령을 둘러싼 공기의 신호에서 배경 소음을 측정합니다. </li><li> [SI 샘플 투자 수익 (ROI)-SI 관리 투자 수익 (ROI)] / SI는 신호 강도가,, [배경 잡음의 SI의 SD]하고 MRI를 대비 대 노이즈 비율 (CNR 계산SD)는 표준 편차는 각 문화 음식값 계산되었다. </li></ol> 5. 대표 결과 체외에서 T2 * 신호 손실 측정은 신호 공극 유물이 생성됩니다 균일한 한천 팬텀과 기포의 부족을 필요로합니다. 이 유물은 SPIO의 정품 T2 * 신호 손실로부터 구분하기가 어렵습니다, 그리고 interpretable 결과에 중요합니다. 공기 유물의 예제뿐만 아니라 정품 철 산화물 T2 * 신호의 손실에 대해 그림 1을 참조하시기 바랍니다. 체외 배양 접시 이미징의 경우, 두께 약 1mm입니다 충분한 축 MRI 조각은, 적절하게 이미지를 전체 요리로 만들 수 있도록, 각 문화 접시에 한천 겔의 적절한 볼륨을 적용합니다. 그림 2는 예상 MRI 신호를 보여줍니다 한천에서 개별 문화 판에서. INT의 영역의 일부 신호 판의 가장자리에서의 변화 때문에 분석은 일반적으로있다erest이 고르지 않은 부분을 방지해야합니다. <img alt="그림 1" src="/files/ftp_upload/3775/3775fig1.jpg" /> 그림 1. ANX-SPIO는 교양 신생아 쥐 cardiomyocytes에서 apoptosis를 감지합니다. 그림 1A는 제어 솔루션 (왼쪽 튜브), ANX-SPIO (중간 튜브) 및 DOX + ANX-SPIO (오른쪽 튜브)로 치료 pelleted 신생아 cardiomyocytes의 사진을 보여줍니다 . DOX의 갈색 색상 + ANX-SPIO 취급 세포, apoptotic 세포의 ANX-SPIO 화합물의 증가 보존을 반영을 적어 둡니다. 체외 MRI 영상에서 그림 1B는 DOX + ANX-SPIO 전지의 강한 T2 * 신호 손실을 보여줍니다. 또한, 공기 방울은 제 4 회 및 신호 ANX-SPIO의 무효, 그리고 CNR의 분석에 영향을 미칠 수와 비슷 5 일 Eppendorf 튜브 (소형 블랙 신호 무효) 사이에 볼 수있다. <img alt="그림 2" src="/files/ftp_upload/3775/3775fig2.jpg" /> 그림 2. ANX-SPIO바인딩은 문화의 apoptotic 세포에 대한 구체적인 것입니다. 그림 2A는 ANX-SPIO의 낮은 농도 (오른쪽 각각 왼쪽)와 ANX-FLUOS 염색법이나 ANX-SPIO의 염색법 중 하나를 받게 문화 요리에서 apoptotic 쥐 신생아 cardiomyocytes를, 보여줍니다. 참고 다른 T2 형광 ANX (ANX-FLUOS) 및 ANX-SPIO 높은 수준의 대비 낮은 수준으로 취급되었다 apoptotic 세포의 * 신호 손실 (흑백 신호). 증가 T2 오른쪽 * 신호 손실이 높은 ASX-SPIO의 농도에 의해 오프 경쟁되었습니다 띄게 감소 ANX-FLUOS 신호와 연관되어 있습니다. 그림 2B는 ANX-SPIO 대 ANX-FLUOS 신호와 분리의 경쟁력 바인딩을 보여줍니다 상수, 나는 K. x 축은 로그 스케일에서 Annexin 단백질 농도를 나타냅니다. 영화 1. <a href="http://www.jove.com/files/ftp_upload/3775/3775movie1.mov" target="_blank">보완을 보려면 여기를 누르십시오</a>영화.

<ol>
	<li>Dash, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+MRI+probe+to+detect+treatment+of+cardiac+apoptosis+in+vivo.">A molecular MRI probe to detect treatment of cardiac apoptosis in vivo.</a> Magn. Reson. Med. , (2011).</li><li>Khym, J. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reaction+of+methylamine+with+periodate-oxidized+adenosine+5'-phosphate.">The reaction of methylamine with periodate-oxidized adenosine 5'-phosphate.</a> Biochemistry. 2, 344-350 (1963).</li><li>Liu, Y. Y., Yu, Y., Zhang, G. B., Tang, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation,+characterization,+and+controlled+release+of+novel+nanoparticles+based+on+MMA/beta-CD+copolymers.">Preparation, characterization, and controlled release of novel nanoparticles based on MMA/beta-CD copolymers.</a> Macromol. Biosci. 7, 1250-1257 (2007).</li><li>Seo, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FeCo/graphitic-shell+nanocrystals+as+advanced+magnetic-resonance-imaging+and+near-infrared+agents.">FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared agents.</a> Nat. Mater. 5, 971-976 (2006).</li><li>Hsiao, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanoparticle+labeling+of+mesenchymal+stem+cells+without+transfection+agent:+cellular+behavior+and+capability+of+detection+with+clinical+1.5+T+magnetic+resonance+at+the+single+cell+level.">Magnetic nanoparticle labeling of mesenchymal stem cells without transfection agent: cellular behavior and capability of detection with clinical 1.5 T magnetic resonance at the single cell level.</a> Magn. Reson. Med. 58, 717-724 (2007).</li><li>Zhao, M., Beauregard, D. A., Loizou, L., Davletov, B., Brindle, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+apoptosis+using+magnetic+resonance+imaging+and+a+targeted+contrast+agent.">Non-invasive detection of apoptosis using magnetic resonance imaging and a targeted contrast agent.</a> Nat. Med. 7, 1241-1244 (2001).</li><li>van Tilborg, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+of+annexin+A5-functionalized+iron+oxide+particles+by+apoptotic+Jurkat+cells.">Internalization of annexin A5-functionalized iron oxide particles by apoptotic Jurkat cells.</a> Contrast Media Mol. Imaging. 4, 24-32 (2009).</li><li>Sosnovik, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+cardiomyocyte+apoptosis+with+a+novel+magneto-optical+nanoparticle.">Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle.</a> Magn. Reson. Med. 54, 718-724 (2005).</li><li>Sosnovik, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+MRI+detects+low+levels+of+cardiomyocyte+apoptosis+in+a+transgenic+model+of+chronic+heart+failure.">Molecular MRI detects low levels of cardiomyocyte apoptosis in a transgenic model of chronic heart failure.</a> Circ. Cardiovasc. Imaging. 2, 468-475 (2009).</li><li>Dumont, E. 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관심의 어떠한 충돌 선언 없습니다.

Annexin V로 SPIO를 링크에 대한 설명 활용 방법은 Annexin의 측면 체인 아민 그룹과 SPIO의 nanoparticle의 카르 복실 moieties을 이용. 구체적인 산화 환원 단계를 통해, 이들 화합물의 공유 결합 결합을 얻을 수 있고, 결과로 작용 nanoparticle을 격리 수 있습니다. 이 방법은 다른 단백질과 관심의 nanoparticles에 일반 수 있습니다. 가장 중요한 단계는 적절한 온도와 부드러운 믹싱으로 수행 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 # </td></tr><tr><td> Superparamagnetic의 철 산화물 </td><td> 오션 나노기술 주식 회사 </td><td> ICK - 40-005 </td></tr><tr><td> Doxorubicin </td><td> 시그마 </td><td> D1515-10MG </td></tr><tr><td> 구분자 SuperMag </td><td> 오션 나노기술 주식 회사 </td><td> N / A </td></tr><tr><td> Zetasizer 나노 DL을 기계 </td><td> Malvern, INC </td><td> N / A </td></tr></tbody></table>

synthesis of an in vivo mri-detectable apoptosis probe

세포 apoptosis는 많은 질병들의 두드러진 특징이며, 질병의 임상 발현은 증거들이 이전에 프로그램된 세포 죽음이 발생합니다. 그것의 초기, 가역 단계에서 apoptosis를 감지하는 수단은 예방이나 치료 조치는 영구적인 손상으로부터 심장을 보호하기 위해 취한 수있는 동안 미리 임상 &#39;창&#39;을 감당할 것입니다. 우리는 본 MRI-감지 대비 에이전트 역할 superparamagnetic 철 산화물 (SPIO) nanoparticles에 avidly apoptosis의 초기, 가역 단계에서 세포에 바인딩 인간 Annexin V (ANX)를, 어원이 같은 말하는 간단하고 강력한 방법을 제시. 활용 방법은 SPIO의 다당류의 껍질에서 카르 복실 그룹을 산화 SPIO의 nanoparticles의 산화로 시작합니다. ANX 단백질 투석을하는 것은 다음 줄이는 버퍼에 SPIO로 ANX의 공유 결합 상호 작용을 촉진하기 위하여 나트륨 borate 솔루션의 설정에 추가됩니다. 나트륨 borohydrid와 최종 감속 단계전자는 감소를 완료하기 위해 수행되는 다음 반응은 침묵하고 있습니다. Unconjugated ANX는 microcentrifuge 여과에 의한 믹스에서 제거됩니다. ANX-SPIO 제품의 크기와 순도는 동적 광 산란 (DL을)에 의해 확인됩니다. 이 방법의 외에도, 또는 수정을 요구하지 않는 등 교차 연결된 철 산화물 입자 활용 방법이나 비오틴-라벨 nanoparticles에 반대 다당류 SPIO 껍질. 따라서이 방법은 관심의 다른 단백질의 활용으로 연장될 수있다 간단 강력한 접근 방식을 나타냅니다.

Watch this Scientific Journal Video about Synthesis of an In vivo MRI-detectable Apoptosis Probe at JoVE.com

Synthesis of an In vivo MRI-detectable Apoptosis Probe

apoptosis의 조기 발견은 질병의 다양한 위험에 처한 세포 인구를 식별할 수 있습니다. 여기 MRI-감지 철 산화물 nanoparticle (SPIO)에 조기 apoptosis 검출 단백질 (Annexin V)를 연결하는 방법을 보여줍니다. 이 방법은 MRI를 - 감지 분자 이미징 프로브를 생성하는 관심의 다른 단백질로 연장될 수있다.

의 합성<em&gt; 생체내에서</em&gt; MRI-감지 Apoptosis 프로브

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

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Synthesis of an In vivo MRI-detectable Apoptosis Probe

9.1K 조회수.  Stanford University Medical Center. apoptosis의 조기 발견은 질병의 다양한 위험에 처한 세포 인구를 식별할 수 있습니다. 여기 MRI-감지 철 산화물 nanoparticle (SPIO)에 조기 apoptosis 검출 단백질 (Annexin V)를 연결하는 방법을 보여줍니다. 이 방법은 MRI를 - 감지 분자 이미징 프로브를 생성하는 관심의 다른 단백질로 연장될 수있다.

비디오: Synthesis of an In vivo MRI-detectable Apoptosis Probe

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

전체 Plasmids의 수제 사이트 감독 돌연변이 유발

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

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생물학

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

유전자 변형으로 인코딩된 칼슘 센서를 사용하여 Apoptosis 동안 Mitochondrial 칼슘 레벨에서 동적 변경 사항 모니터링

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

시토크롬의 세포질 Microinjection 의한 Apoptosis의 활성화 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

모자이크 성인의 라이브 이미징 토마토 / GFP-FLP / FRT 방법<em&gt; 초파리</em&gt; 광 수용체 (photoreceptor)

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

생체</em에서 단백질 복합체를 분리> 가교 접근 초파리 태아

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

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 ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

<em&gt; 예 생체 내</em&gt; 문화 시스템 갑상선 개발을 연구하는

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polysom​​e 프로파일 링 포유 동물 세포에 선택적 mRNA의 번역 평가

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...