우리는이 작업에 설명 된 프로토콜에 대한 전문 기술 지원을 르네 L. Pasker 및 Harpreet K. Brar에게 감사의 말씀을 전합니다. 또한, 우리는 우리에게 시간을 허용 자신의 실험실 구성원의 지원과 함께, 위스콘신 - 매디슨 대학의 듀크 대학 앨런 D. Attie 크리스토퍼 B. Newgard의 멘토링을 인정하고 최적화하기 위해 필요한 지원하고자하는 설명 프로토콜. 특히, 우리는 생산적인 토론과 조언을 Attie 연구소의 한스 Hohmeier, Danhong 루, 헬레나 윈필드 Newgard 연구소의 마리아 Rabaglia 감사합니다. 이 작품은 NIH 부여 DK080845 및 청소년 당뇨병 594에 의해 지원되었다 (MEK에 대한) 연구 재단 보조금 17-2011-608

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저자가 공개하는 게 없다.

세계 인구의 19.5 %에 영향을 미칠 것으로 예상 당뇨병의 유병율로, 신규의 연구 기술의 요구 사항을 모두 이해하고 18 당뇨병을 치료하는 데 필수적이다. 본 섬 격리는 시험관 실험에 사용되는 잘 확립 된 프로토콜이며, 약간의 수정을 11,14,16으로 이전에 제시되어 있습니다. 인슐린 분비는 캠프와 같은 업스트림 구성 요소에 초점을 맞추고, 고립 된 섬에 대한 일반적인 다운 ?...

euglycemia의 엄격한 관리는 조절되지 않는 제 1 형과 제 2 형 당뇨병 1의 병리의 모든 특징입니다과 같은 신경 병증, 신장 병증, 망막 병증과 같은 합병증을 방지하기 위해 필수적입니다. 제 1 형 및 2 형 당뇨병을 모두 감소 β-세포 기능 및 질량은 혈당 농도를 2 교란. 인슐린을 생산하는 β-세포, 손상된 β-세포의 인슐린 분비 및 형식 주변 인슐린 신호 2 형 당뇨병의 엄청난 손실 면역 매개 성 제 1 형 당뇨병의 결과를 함께 고혈당, 이상 지질 혈증, 결국 결과 증가 간 포도당 생산을 촉진하는 반면 각각의 β-세포 3 β-세포 질량과 인슐린 분비 능력의 상실. 모두 제 1 형 및 2 형 당뇨병의 진행에 기본 β-세포의 메커니즘을 이해하는 것은 희망이 질병을 예방하고 치료하는 새로운 치료로 상승을 줄 것이다. 체외 TI의그러한 INS-1과 같은 MIN6 ssue 배양 모델은, β-세포 라인을 불멸화 특정 β-세포의 기능을 이해하는데 유용한 도구가 될 수있다. 그러나, 베이 내의 상이한 세포 유형 간의 상호 작용 자체는 β-세포의 기능을 조절할 수있다. 예를 들어, 글루카곤의 주변 분비 영향 (α-세포에서 방출)과 인슐린 분비를 증가 및 감소에 소마토스타틴은 (δ-세포에서 방출), 각각 내분비 응답 4 셀 전지 근접의 중요성을 보여줍니다. 또한, β-세포 사이의 간극 결합 인슐린 5의 출시를 주다. 진보는 더 나은 포도당 고립 된 섬의 생리적 반응을 복제 인슐린 라인을 생성하는되었습니다 있지만 또한, (예를 들어, INS-1 유래 13분의 832 및 3분의 832 세포주), 자신의 포도당 응답은 여전히 정상 쥐와 다르다 6,7 섬. 또한, 이들 클론 인슐린 종 세포주 응답글루카곤 유사 펩타이드 -1 (GLP-1)의 효능을 서로 극적 다를뿐만 아니라, 통상의 아일렛 6 개있다. 따라서, 불후의 세포 라인 에이전트에게 캠프의 생산에 영향을 미치는 시금에 가장 적합한 모델을 대표하지 않을 수 있습니다. 인슐린 유래 세포 라인과는 대조적으로, 전체 동물 모델에서 유일하게 β-세포의 기능을 연구하는 것은 합병증의 자신의 세트를 제공합니다. 내분비 조직 작업에서 가장 큰 과제 중 하나는 방출 호르몬의 정확한 농도를 측정한다. 구체적으로, 간은 인슐린 대사에 중요한 역할을하고, 췌장의 혈류가 간으로 직접 진행한다. 따라서, 혈장 인슐린 둘레 정확하게 췌장 자체 또는 인슐린 분비 (8)의 속도에 다른 처리의 영향에서 분비되는 인슐린의 양을 묘사 할 수 없습니다. 또한, 글루카곤의 신 대사는 섬의 α-세포에서 글루카곤 9 출력의 신뢰성을 제한 할 수 있습니다. 따라서, 생체 외 실험에 대한 기본 마우스 섬을 분리하는 것은 섬이 생체 내에서 만들어 측정을 보완하기 위해 특정 자극에 응답하는 방식에 대한 정확한 이해를 제공합니다. 마우스 섬의 분리를위한 의정서는 (성공을 증가하는 데 도움이 될 수 있습니다 약간의 수정) 그룹의 수 (10, 11)에 의해 사용되는 잘 확립 된 프로토콜입니다. 또한, cAMP의 생산의 결정은 β-세포의 인크 레틴 반응성 직접 판독을 허용한다. 캠프 측정, 단백질 함량 및 인슐린 분비와 함께 또한 β-세포 기능에 결함이 캠프 10 근위부 또는 원위부 놓여 있는지 여부를 확인하는 데 도움이 같은 캠프 샘플 준비에서 정량화 할 수있다. 이 프로토콜의 마지막 캠프의 내용과 인슐린 분비 응용 프로그램이 다른 가운데, 제약 및식이 성분의 영향을 이해하기위한 매우 강력한 도구가 될 수 있습니다의, 캠프 및 인슐린 분비에. 단독 포도당의 자극 이외에, 다른 화합물이 캠프 및 인슐린 분비 10,11 변화를 측정하는데 사용될 수있다. 인슐린은 우리가 절연 아일렛 같은 글루카곤 및 소마토스타틴 같은 다른 호르몬,뿐만 아니라 사이토 카인, 에이코 사 노이드, 및 사이 클릭 아데노신 모노 포스페이트 것으로 분석 차 호르몬이지만 마지막으로, 또한 과도 자극 분석 또는 정량함으로써 역시 측정 할 수있다 문화 매체 (12)에서 자신의 수준. 마지막으로, 다른 많은 다운 스트림 애플리케이션 등 섬 이식, PCR이나 마이크로 어레이 분석 정량 실시간에 대한 RNA 분리, 단백질로, 추구 할 수 있도록 외부에서이 원고의 범위, 기술 콜라게나 분리 방법 섬 격리 섬 보존 할 수 있지만, 웨스턴 블로 팅, 섬 매립 및 면역 형광 이미징 및 섬 세포 repli의 척도로서 [3H] - 티미 딘 설립을위한 격리이전 조브 기사 13-16에서 설명 된 일부 양이온. 전반적으로, 프로토콜에 설명 된 섬 격리 절차에 따라 치료법을 개발 및 β-세포의 기능을 강화하기위한 신약 개발을 촉진하기위한 중요하고 유용한 정보를 연구자를 제공 할 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="502">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Collagenase: Collagenase from Clostridium histolyticum suitable for isolating active islets</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C7657</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Ficoll 400</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>F9378</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hanks Balanced Salt Solution 10X</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td>14065-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hepes</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>H3375</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">RPMI 1640 (powder)</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">31800-022</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>A7888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3/0 Silk Suture Thread</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>18020-30</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dumont #5 Forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>11251-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">0.8 mm Forceps&#160;&#160;</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">11050-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Curved Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14061-10</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">15003-08</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dissecting Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14002-14</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">5ml BD Luer-Lok Syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309646</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">1ml BD syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309628</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">30 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305106</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">27 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305109</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sharpening Stone</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">29008-01</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-2-2 tribromoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">T48402-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-methyl-2-butanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>240486-100mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sodium Chloride (NaCl)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Potassium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>P3911</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:235px;">Monopotassium Phosphate (KH2PO4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0662</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Sodium Bicarbonate (NaCHO3)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">S6014</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2 *2H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C3881</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4 *7H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">M9397</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">15140-122</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:235px;">Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:136px;">SH30088.03HI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">5879-100MG</td>
		</tr>
	</tbody>
</table>

a method for mouse pancreatic islet isolation and intracellular camp determination

조절되지 않는 혈당은 당뇨병의 특징이며, 신경 병증, 신장 병증, 망막 병증과 같은 합병증을 촉진합니다. 당뇨병, 면역 매개 형 1과 비만 연결된 제 2 형 모두, 당뇨병의 병태 생리 및 치료 메커니즘을 서술하기위한 연구의 증가 유행으로 매우 중요하다. 췌장의 랑게르한스 섬의 β-세포가 적절하게 혈중 포도당 농도에 반응하여 인슐린을 분비하는 책임이 있습니다. 포도당 및 다른 영양소는, β-세포가 특정 호르몬에 의해 자극 또한, 세포 내 환상 아데노신 모노 포스페이트의 생산을 증가 β-세포 수용체의 식사 행동에 대한 응답으로 창자에서 분비되는 인크 레틴을, (라고 캠프). 감소 β-세포 기능, 대용량 및 인크 레틴 응답은 2 형 당뇨병의 병태 생리에 기여하는 잘 이해하고, 또한 점점 위스콘신 연결되고있다일 1 형 당뇨병. 본 마우스 섬 격리 및 캠프 결정 프로토콜은 질병의 진행 및 치료 적 개입, 세포 내 cAMP의 생산의 조절을 통해 행동 수용체를 인크 레틴 수용체에 의해 매개 또는 관련되는 특히 증진 메커니즘을 묘사하는 데 도움이 도구가 될 수 있습니다. 전용의 cAMP 측정이 설명 될 것이지만, 기술 된 아일렛 분리 프로토콜은 포도당, 인슐린 분비, [3 H] - 티미 딘 혼입 단백질 풍부하고, mRNA 발현을 자극 포함한 다른 많은 다운 스트림 애플리케이션을 허용 깨끗한 제제를 생성한다.

격리 기간 동안 높은 섬 수익률을 보장하기 위해, 프로토콜에 설명 된 수술 기법을주의 깊게 추적 관찰을해야한다. 여기에 제시된 기술은 각 실험실에 맞게됩니다 만, 성공적인 분리로 이어질 것입니다 몇 가지 중요한 단계가 있습니다. 담관이 쉽게 접근 할 수 있도록하기 위해서는, 장기가 마우스의 오른쪽 (도 1)로 변위하는 것을 추천합니다. 더욱이, 이러한 제한은 적은 무게가 팽?...

Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

랑게르한스의 고립 된 마우스 독도를 이용하여 체외 β-세포 기능을 시금 당뇨병의 병태 생리 및 치료의 연구에 중요한 구성 요소입니다. 많은 다운 스트림 응용 프로그램을 사용할 수 있지만,이 프로토콜은 특히 β-세포의 기능을 결정하는 중요한 매개 변수로 세포 내 순환 아데노신 monophosphate (캠프)의 측정에 대해 설명합니다.

마우스 췌장 섬의 분리 및 세포 내 cAMP를 결정하는 방법

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

a-method-for-mouse-pancreatic-islet-isolation-intracellular-camp

Research

JoVE Journal

Biology

64.9K 조회수.  University of Wisconsin-Madison.  랑게르한스의 고립 된 마우스 독도를 이용하여 체외 β-세포 기능을 시금 당뇨병의 병태 생리 및 치료의 연구에 중요한 구성 요소입니다. 많은 다운 스트림 응용 프로그램을 사용할 수 있지만,이 프로토콜은 특히 β-세포의 기능을 결정하는 중요한 매개 변수로 세포 내 순환 아데노신 monophosphate (캠프)의 측정에 대해 설명합니다. 

비디오: A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Murine Pancreatic Islet Isolation

Murine 췌장 아일릿 격리

연구

생물학

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

인간 췌장 아일릿 격리 : 부품 I : 소화 및 췌장 조직의 수집

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37&deg;C overnight culture before islets were transplanted or used for research.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

인간 췌장 아일릿 절연 : 2 부 : 인간 아일 레츠의 정화와 문화

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

컴퓨터를 이용한 대규모 시각화 및 췌장 아일릿 질량, 크기 유통과 건축의 부량

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Langerhans 절연의 마우스 작은 섬은 정화의 Collagenase와 중성 단백 분해 효소의 조합을 사용하여

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

마우스 기본 췌장 포상 세포의 분리 및 문화

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

근적외선 (NIR) 빛은 마우스 전정 감각 상피 세포에서 미토콘드리아 기능의 마커의 발현을 증가

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

신생아 및 성인 마우스 피부의 1 차 마우스 각질 세포의 분리 및 배양

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.