이 작품은 NIH (PTF까지)에서 부여 DK078732에 의해 지원되었다.

1. 쥐 Islets의 Adenoviral 형질 도입 및 Culturing <ol><li> 의 필요한 번호 미디어 2 ML (8 MM 포도당 10 % 태아 소 혈청, 50 단위 / ML 페니실린, 50 μg / ML의 스트렙토 마이신을 포함하는 RPMI 1640 미디어)를 추가하여 6 개 잘 아닌 조직 문화 코팅 플레이트를 준비 웰스. 노 바이러스 제어를위한 한 대씩, 바이러스 컨트롤 (예 : GFP-표현 아데노 바이러스)과 실험 집단 - 예를 들어, 일반적인 실험은 세 우물이 필요할 수 있습니다. </li><li> 적어도 30 분 동안 조직 문화 인큐베이터에 그것을 배치하여 37 ° C로 접시를 따뜻하게. </li><li> 바로 6 - 잘 아닌 조직 문화 코팅 플레이트의 개별 우물에 쥐 섬 절연 18,19, 장소 100-200 islets에 따라. 육십 islets은 인슐린 분비 및 thymidine의 정관 assays가 필요합니다. 남은 islets는 유전자 발현 연구 또는 immunoblotting을위한 단백질 분리를위한 RNA 분리에 사용할 수 있습니다. </li></ol> [참고 :이 시점부터, biohazardous 재료의 취급, 사용 및 처분을위한 제도적 절차를 따르시기 바랍니다.] <ol start="4"><li> 부드럽게 소용돌이도의 중심에 islets를 가져올 수있는 판. </li><li> 직접 접시의 중앙에 islets에 아데노 바이러스를 피펫. 감염 100-500 multiplicities을 (입니다만, 바이러스성 상패 - 성형 단위로 목표 세포의 비율)를 사용합니다. </li><li> 5 분 동안 islets을 방치 했죠. </li><li> 조직 문화 인큐베이터 (37 ° C에서 5 % CO 2)의 접시를 놓습니다. </li><li> 24 H 후, 부드럽게 소용돌이 우물의 센터로 islets 가지고 잘 신선한 매체를 포함하는 새에 P200 micropipette를 사용 islets를 전송하는 판. islets가 접시에 첨부되어있다면, 그들은 부드럽게 피펫 팁과 dislodged 수 있습니다. </li></ol> [주 : 적절한 형질 도입의 efficien을 확인하려면islets 그때 섬 핵심으로 아데노 바이러스의 침투를 확인하기 위해 공촛점 현미경을 통해 몇 군데 할 수있는 싸이, 제어 바이러스 표현 GFP의 사용이 유용합니다.] <ol start="9"><li> 최적화 파일럿 연구에서 실험 원하는 타이밍에 따라 추가로 24-72 H에 대한 문화 islets. 예를 들어, proliferative 반응의 유도는 24-72에서 H 또는 관심있는 유전자의 최저 48 또는 72 시간 요구할 수 이르기까지 시간이 필요할 수 있습니다. 새로운 미디어에 매일에 islets를 전송합니다. </li><li> 실험 1을 포함하는 미디어 문화 islets을의 마지막 24 H 들어 μCi [메틸-3 H] -thymidine/ml 매체 (일반적으로 1 μl thymidine / ML 미디어). </li></ol> [참고 :이 시점부터, 방사성 물질의 취급, 사용 및 처분을위한 제도적 절차를 따르시기 바랍니다.] 2. 인슐린 분비 분석 <ol><li> 준비분비 분석 완충액 (SAB) 10X 주식 용액 (1.14 M NaCl, 47 MM KCl, 12 MM KH 2 PO 4, 11.6 MM MgSO 4)와 CaCl 2 100X 주식 용액 (0.25 M CaCl 2). 이러한 주식 솔루션은 미리 준비하고 상온에서 저장할 수 있습니다. </li><li> 갓에서 작동 SAB (10X SAB, 1 M의 HEPES, 100X CaCl 2 0.5 ML 35 % BSA, 0.11 g NaHCO 3의 0.28 ML 1 ML 및 멸균 물 50 ML의 5 ML)의 50 ML 준비 50-ML 튜브 원뿔 따뜻한 37에 ° C ~ 37 ° C waterbath에 배치하여. </li><li> 피펫 10 15 ML 원뿔 튜브로 작업 SAB의 ML하고 높은 포도당 (16.7 ㎜) SAB를 준비하기 위해 2.5 M D-글루코오스의 66.8 μl를 추가합니다. </li><li> 낮은 포도당 (2.8 ㎜) SAB를 준비하는 작업 SAB의 나머지 40 ML에 2.5 M D-글루코오스의 44.8 μl를 추가합니다. </li><li> 라벨 세 1.7-ML microcentrifuge의 6 잘 플레이트의 각 잘위한 튜브하고 인산 버퍼 식염수 (PBS) 1 ML을 추가합니다. </li></oL&gt; [주 : islets는 방사성들에 따라, 방사성 물질의 취급, 사용 및 처분을위한 제도적 절차를 따르시기 바랍니다.] <ol start="6"><li> 각 microcentrifuge 튜브로 20 islets를 놓습니다. 각 microcentrifuge 튜브에 comparably 크기의 islets을 추가하는 모든 시도를 확인하십시오. 예를 들어, 각각의 튜브는 5 소형, 10 중간, 5 대형 islets합니다 (그림 1 참조)가 포함될 수 있습니다. </li></ol> [주 : Islets는 해부 입체경 또는 표준 현미경을 사용하여 시각하실 수 있습니다.] <ol start="7"><li> islets 중력 (~ 2 분)에 의해 튜브의 바닥에 정착 후 micropipette으로 PBS를 대기음 및 폐기. </li></ol> [주 : 중력에 의해 침강에 대한 대안으로 튜브는 1 분 300 XG에 centrifuged 수 있습니다.] <ol start="8"><li> 사전 배양은 낮은 GLU 400 μl를 추가할편안히 앉다 SAB는 조직 문화 인큐베이터 (37 ° C에서 5 % CO 2), 60 분 미리 품어으로 튜브를 (그들의 뚜껑 열린 포함) 넣습니다. 사전 인큐베이션 낮은 포도당 SAB 및 폐기를 기음. </li><li> 기저 인슐린 분비의 경우 낮은 포도당 SAB 400 μl를 추가, 조직 문화 인큐베이터 (37 ° C에서 5 % CO 2)로 튜브를 (그들의 뚜껑 열고 사용) 배치하고, 60 분 동안 품어. 낮은 포도당 SAB를 수집하고 인슐린 radioimmunoassay 저장. </li><li> 자극 인슐린 분비의 경우 높은 포도당 SAB 400 μl를 추가, 조직 문화 인큐베이터 (37 ° C에서 5 % CO 2)로 튜브를 (그들의 뚜껑 열고 사용) 배치하고, 60 분 동안 품어. 높은 포도당 SAB를 수집하고 인슐린 radioimmunoassay 저장. </li></ol> 3. Thymidine의 설립 분석 <ol><li> 한 ML PBS를 추가, islets 중력에 의한 튜브의 바닥에 정착 후, micropipette으로 PBS를 대기음 폐기하고, 이것을 반복한 단계. </li><li> 500 μl 얼음 추위 trichloroacetic 산 (TCA 10 % W / V)를 추가하고 30 분 동안 얼음에 품어. </li><li> 4에 3 분 ° C. 16 000 XG에 튜브를 원심 분리기 </li><li> TCA를 대기음, 0.3 N NaOH 80 μl를 추가하고 실온에서 30 분 동안 품어. 이 기간 동안 5-10 s에 대한 노골적인 와동 샘플을 매 10 분. </li><li> 7 ML 액체 섬광 계수 튜브로 이코노 안전 계수 칵테일 4 ML을 추가합니다. </li><li> 섬광 계수 튜브에 시료 50 μl를 추가, 튜브를 쏠 잠깐 흔들 및 액체 신틸레이션 계수기로 계산됩니다. </li><li> 제조 업체의 프로토콜에 따라 bicinchoninic 산성 (BCA) 분석 및 시료 10 μl를 사용하여 단백질 농도를 측정합니다. </li></ol> 4. 데이터 분석 <ol><li> 제조 업체의 프로토콜에 따라 인슐린 radioimmunoassay를 수행합니다. </li><li> 단백질 conc로 인슐린 분비 및 thymidine의 설립 데이터를 정규화entration. </li></ol> 5. 대표 결과 쥐 islets의 섬 복제 및 베타 세포 기능을 평가하기위한 실험의 예는 그림 2에 표시됩니다. 이 예제는 robustly 베타 세포 기능을 변경하지 않고도 섬 복제를 자극 가상 &quot;# 6 진&quot;의 adenoviral overexpression를 보여줍니다. 상단 패널에서 thymidine 결합 분석의 결과는 &#39;진 # 6 &quot;의 표현을 증가하는 등 thymidine의 결합에 의해 측정의 DNA 합성을 증가 것을 보여줍니다. 쥐 섬에있는 세포의 대부분은 베타 세포이기 때문에, 그것은 thymidine의 설립이 증가 베타 세포 복제의 증가를 나타내는 가능성이 높습니다. 그러나 확실한 실험은 단단히 이것을 수립 실시되어야합니다. 하단 패널에서 인슐린 분비 분석의 결과는 &#39;진 # 6 &quot;나, 즉, 기본 베타 세포 기능 중 하나를 변경하지 않은의 overexpression을 보여주낮은 높은 포도당에서 nsulin 분비. adenoviruses과 치료에 따라 islets의 섬 격리와 건강의 품질이 낮은 높은 포도당 농도의 인슐린 분비의 배 증가로 표시됩니다. &#39;진 # 6 &quot;장애인 베타 세포 기능의 표현을 증가하는 경우, 이것은 가능성이 높은 stimulatory 포도당 농도 (16.7 ㎜)에서 분비 인슐린의 감소로 반영됩니다. 포도당 농도를 변화를위한 선량 - 반응 곡선도 수행할 수 있습니다. <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="그림 1"/> 그림 1. 유전자 발현의 adenoviral-매개 변경에 따라 분리된 쥐의 islets에 섬 복제 및 베타 세포 기능을 평가하기위한 프로토콜의 개요. 갓 고립된 쥐 islets 24 H 위해 adenoviruses에 노출된 후 96 H까지 배양해 있습니다. Thymidine의 설립이에서 인슐린 분비의 측정 다음, 마지막 24 H로 평가된다낮은 높은 포도당. <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="그림 2"/> 그림 2. 제어 아데노 바이러스와 아데노 바이러스 overexpressing &#39;진 # 6 &quot;으로 표시된 가상의 유전자를 사용하여 실험의 결과. 상단 패널 thymidine의 설립과 하단 패널 인슐린 분비를 보여줍니다.

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Chem. 270, 1295-1299 (1995).</li><li>Szot, G., Koudria, P., Bluestone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Pancreatic+Islet+Isolation.">Murine Pancreatic Islet Isolation.</a> J. Vis. Exp. (7), e255 (2007).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

복제를 자극하고 베타 세포의 기능을 향상시키기 위해 변조된 수 수립 경로는 당뇨병의 두 주요 형태와 관련이 있습니다. 기능적인 베타 세포 질량이 determinants을 평가, 인슐린 - secreting 세포의 존재와 기능에 종속되어 있기 때문에 동시에 그것의 장점이 있습니다. 이 프로토콜은 단백질의 overexpression 또는 억제 그러면 생체내에 효능에 대해 테스트할 수 체외에서 기능적인 베타 세포...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> RPMI 1640 미디어 </td><td> Gibco </td><td> 11,879 </td><td></td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> Gibco </td><td> 15,140 </td><td></td></tr><tr><td> 6 - 잘 플레이트 </td><td> BD-팔콘 </td><td> 35-1146 </td><td> 비 TC 처리 </td></tr><tr><td> [메틸-3 H]-thymidine </td><td> Perkin 엘머 </td><td> NET027Z001MC </td><td> 한 MCI / ML </td></tr><tr><td> 마이크로 원심 분리기 튜브 </td><td> Denville </td><td> C2170 </td><td> 1.7 ML </td></tr><tr><td> NaCl </td><td> 시그마 </td><td> 59,888 </td><td></td></tr><tr><td> KCl </td><td> Acros </td><td> 42,409 </td><td></td></tr><tr><td> KH 2 &lt;/ 서브&gt; PO 4 </td><td> Acros </td><td> 20,592 </td><td></td></tr><tr><td> MgSO 4 </td><td> Acros </td><td> 41,348 </td><td></td></tr><tr><td> CaCl 2 </td><td> Acros </td><td> 34,961 </td><td></td></tr><tr><td> HEPES </td><td> 시그마 </td><td> H0887 </td><td> 1 M 솔루션 </td></tr><tr><td> 35% BSA </td><td> 시그마 </td><td> A7979 </td><td></td></tr><tr><td> NaHCO 3 </td><td> Acros </td><td> 42,427 </td><td></td></tr><tr><td> D-글루코오스 </td><td> 시그마 </td><td> G8769 </td><td></td></tr><tr><td> TCA </td><td> 피셔 과학 </td><td> SA9410-1 </td><td> 10% W / V </td></tr><tr><td> NaOH </td><td> Acros </td><td> 12,426 </td><td></td></tr><tr><td> 섬광은 튜브를 센다 </td><td> Sarstedt </td><td> 58.536 </td><td> 7 ML, PP </td&gt; </tr><tr><td> 섬광 계수 튜브 캡 </td><td> Sarstedt </td><td> 65.816 </td><td></td></tr><tr><td> 이코노 - 안전 계수 칵테일 </td><td> RPI </td><td> 111,175 </td><td></td></tr><tr><td> 인슐린 RIA </td><td> 지멘스 </td><td> TKIN2 </td><td></td></tr><tr><td> BCA 분석 키트 </td><td> 써모 과학 </td><td> 23,250 </td><td></td></tr><tr><td></td><td></td><td></td><td> 장비 </td></tr><tr><td> 원심 분리기 </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> 섬광은 튜브 랙을 계산 </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> 액체 섬광 계수기 </td><td> Perkin 엘머 </td><td> 트라이 수화물 2910TR </td><td></td></tr></tbody></table>

assessing replication and beta cell function in adenovirally-transduced isolated rodent islets

포도당 항상성은 주로 각각 췌장 베타와 알파 세포에서 분비 내분비 호르몬의 인슐린과 글루카곤에 의해 제어됩니다. 기능성 베타 세포 질량은 해부학 베타 세포 질량뿐만 아니라 영양 하중에 대응하는 베타 세포의 능력에 의해 결정됩니다. 기능적인 베타 세포 대량의 손실은 당뇨병 1-3의 두 주요 형태의 핵심이다. 제 1 형 당뇨병의 면역 공격의 감소 기능성 베타 세포 질량 결과 반해, 제 2 형 당뇨병에이 감소가 적절하게 인슐린을 분비하는 베타 세포의 무능력과 메커니즘의 간부로부터 베타 세포의 파괴 양쪽에서 개발하고 있습니다. 따라서 기능적인 베타 세포 질량을 복원하기위한 노력은보다 나은 치료 및 당뇨병에 대한 잠재적인 치료에 답해야합니다. 노력은 복제를 자극하고 베타 세포의 기능을 향상시키기 위해 악용될 수있는 분자 경로를 식별하는 진행되고 있습니다.이상적인 치료 목표는 베타 세포의 성장과 기능을 모두 향상시킬 것입니다. 하지만 아마도 더 중요한 베타 세포 성장을 자극 전략은 혼란 베타 세포 기능 (예 : 일부 oncogenes과 등)와 그 반대의 비용으로 오는지를 파악하는 것입니다. 고립된 쥐 islets에서 대상 유전자의 체계적 억제하거나 overexpressing 표현함으로써, 하나는 증가 기능성 베타 세포 대량 4-6에 대한 잠재적인 치료 표적을 식별할 수 있습니다. Adenoviral 벡터는 고립된 쥐 islets 4,7-15의 효율적 overexpress 또는 최저의 단백질로 근무하실 수 있습니다. 여기서는 adenoviral 형질 도입을 이용한 유전자 발현을 조작하고 고립된 쥐 islets (그림 1)의 섬 복제 및 베타 세포 기능을 평가하기위한 방법을 제시한다. 이 메소드는 베타 세포 복제 또는 기능 5,6,8,9,16,17 보죠 소설 대상을 식별하기 위해 이전에 사용되었습니다.

Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

이 프로토콜은 하나가 당뇨병 치료를위한 잠재적인 치료 표적을 찾기 위해 기능성 베타 세포 질량을 조절 요소를 식별할 수 있습니다. 프로토콜은 adenoviruses과 유전자 발현의 조작에 따라 분리된 쥐의 islets에 섬 복제 및 베타 세포 기능을 평가하기위한 간소화된 방법으로 구성되어 있습니다.

에서 복제와 베타 세포 기능을 평가 Adenovirally-transduced 절연 쥐 Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

assessing-replication-beta-cell-function-adenovirally-transduced

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Medicine

13.9K 조회수.  Indiana University School of Medicine. 이 프로토콜은 하나가 당뇨병 치료를위한 잠재적인 치료 표적을 찾기 위해 기능성 베타 세포 질량을 조절 요소를 식별할 수 있습니다. 프로토콜은 adenoviruses과 유전자 발현의 조작에 따라 분리된 쥐의 islets에 섬 복제 및 베타 세포 기능을 평가하기위한 간소화된 방법으로 구성되어 있습니다.

비디오: Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

를 사용하여 절연 마우스 마음에 심장 기능 및 에너 지학 평가 31 P NMR 분광학

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

연구

의학

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

엔도 - PAT 2000 내피 Vasodilator 기능을 평가

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

절연 골격근의 묘 - 기계 분석

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

부분 Pancreatectomized 환자에서 인간 아일 레츠의 분리

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

인간의 췌장 Islets위한 염색법 프로토콜

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; DB</sup&gt; 2 형 당뇨병의 마우스 모델 : 본래 독도 췌장 섬 세포의 분리 및 라이브 셀 2 광자 이미징

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

높은 콘텐츠<em&gt; 체외</em&gt; 췌장 작은 섬의 β 세포 복제 디스커버리 플랫폼

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

양적 3D<em&gt; 인 실리코</em알츠하이머 병의 쥐 모델에서 뇌 아밀로이드 - 베타 탐식&gt; 모델링 (q3DISM)

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

여성 작은 설치류에 신경을 살려주는 중간 요도 폐색 (네모)

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal 이식 마우스 모델에서 췌 장 독도의

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...