저자는 UCSF 자원 할당 프로그램이 제공하는 자금을 인정하고 싶습니다.

모든 동물의 작업은 국립 보건원 (National Institutes of Health)의 실험 동물의 관리 및 사용을위한 설명서의 권장 사항을 엄격히 준수하에 수행되었다. 이 프로토콜은 캘리포니아 대학의 기관 동물 관리 및 사용위원회 (IACUC), 샌프란시스코에 의해 승인되었습니다. 1. 마우스 배아 눈물샘 (LG) : 수확 및 이외에 Microdissection <ol><li> , 과학 연구 윤리위원회의 기관 동물의 승인 절차를 수행하면 해당 배아 일에 수확 배아 자궁 전위에 의해 확인 다음, 상승 CO 2 흡입 임신 CD-1 (또는 유전자 변형) 여성을 초과 안락사. 질 플러그 검색 E0의 날을 지정합니다. 참고 : 눈물샘 (LGS)은 이후 E14 하나의 꽃 봉오리 단계에서 수확 할 수있다. 그러나, E16 (5-10 싹) 배아는 체외 문화에 대한 가장 일관된 결과를 제공합니다. </li><li> V를 소독70 % 에탄올을 사용하여 마우스의 측면 entral. 피부를 조이고 무균 수술 가위로 중간 선에서 절개를; 피부의 실수로 복강을 노출 복막. 100 U / ㎖ 페니실린과 100 μg의가 / ㎖ 스트렙토 마이신이 보충 된 각각의 자궁 뿔과 장소 차가운 PBS에서 (인산염 완충 식염수) 또는 DMEM의 F12 미디어의 상단에있는 mesometrium을 따라 절단하여 2 자궁 뿔을 제거합니다. </li><li> 집게를 사용하면 (# 5), 양막 낭에서 배아를 제거하고 차가운 PBS를 포함하는 두 번째 멸균 페트리 접시에 놓습니다. 눈이나 머리 부분을 만지지 않도록주의하십시오. </li><li> transillumination 기지와 해부 현미경에 배아를 놓습니다. 1.7 - 배아 &lt;E17 경우, 단계 1.5을 수행합니다. 배아 경우&gt; E17는 다음과 같이 따라서 1.8 단계로 진행, 도움이 될하지 않고 반드시 할 수있다. </li><li> 바로 아랫 부리 아래의 몸통 (단계 1, 그림 2) 날기에서 머리를 절단GA 멸균 메스 (# 10 블레이드). 하악이 해부 접시에 지금이되도록 머리를 돌립니다. 머리와 약 ¼ 뇌 (2 단계, 1 차 컷, 그림 2)의 등 쪽을 제거하는 메스를 안정시키기 위해 집게를 사용하여 -이 연골 E15 주위에 경직되면 특히 중요합니다. </li><li> 메스는 눈 사이의 코를 관통 할 수 있도록 머리의 방향을. 눈은 별도의 절반 (2 단계 2 차 컷, 그림 2)에 지금 그래서, 헤드의 중심을 잘라주십시오. </li><li> 두 # 5 집게 사용하여 머리의 절반을 과잉 조직 (그림 2 단계 3 참조)를 제거합니다. (눈의 낮은 후방 모서리에 위치) LG는이 제거 과정에서 손실되지 않도록 머리를 됐는지 확인하십시오. 초과 뇌, 연골, 비강 조직 주변과 눈 아래를 제거합니다. 선이 시점에서 거의 보이지 때문에, 적절히 과량 조직 수거 t 후에 배향 눈을 보장O LG의 위치를​​ 잃지 마십시오. </li><li> 조심스럽게 뒤, 두 집게로 눈의 하단으로부터 피부를 잡아. 조심스럽게 LG를 노출 할 수있는 집게로 열린 당겨 피부를 제거합니다. LG를 시각화하기 위해 조직 위와 아래에 추가 조명을 사용합니다. 주 : LG 전자, 어두운, 압축 된 중간 엽 내 덕트에 꽃 봉오리 같은 모양으로 구별이 시점에서 볼 수 있어야합니다. </li><li> 조심스럽게 LG 및 관련 중간 엽를 LG 또는 관련 덕트를 잡아 않도록주의하면서, 집게를 사용하여 거리 주변 조직에서 (다이어그램 및 이미지 참조) 해부하기 시작합니다. 글 랜드는 주변 조직에서 무료로되면, 부드럽게 LG 덕트의 바닥에있는 눈을 둘러싸고있는 상피 세포를 양손으로 전체 선을 제거합니다. 압축 된 중간 엽에 상피 invaginates으로 관찰 할 수있는 상피 세포를 둘러싸고있는 중간 엽을 유지하기 위해주의하십시오. 참고 : tissu의 일부 추가 제거즉 (임의의 작은 뼈 조각, 근육, 및 결합 조직) 조직 이웃으로부터 / 성장 인자 시그널링을 피하기 위해 필요하다. </li><li> 문화, 수확 4 - 5 땀샘과 판 당 관련 중간 엽 (LGS 즉시 해부에 도금) RT-PCR 6 RNA 용해 버퍼 100 ㎕ 당 14 땀샘의 최소를 수집합니다. </li></ol> 2. 생체 내 문화 플레이트를 준비 이 절차는 현상 침샘 7,8에 대해 사용 된 것과에 기초한다. <ol><li> 바닥이 유리로 된 50mm 직경의 마이크로 웰 요리 (7) 50 ㎍ / ml의 아스코르브 산 50 ㎍ / ㎖의 홀로 트랜스페린과 문화 미디어 (100 U / ㎖ 페니실린 및 100 ㎍ / ml의 스트렙토 마이신으로 보충) DMEM의 F12 200 μl를 추가합니다. 참고 : 우리가 이전에 배아 타액선의 ​​형태 형성을 극대화하기 위해 그것을 발견 DMEM의 F12가 선택되었다. </li><li> 13mm의 POLYCARB 플로트미디어의 상단에 0.1 μm의 기공 네이트 멤브레인 필터. </li><li> 선택적 단계가 : 3 mg / ml의 최종 농도를 만들어 DMEM / F12와 1 (부드럽게 섞어 200 μL 피펫을 사용하여, 원심 분리를 1 분 동안 기포가 나타나는 경우) : 3-D 라미닌 1 희석. 추가이 15 μL 필터하기 위해 3-D의 라미닌을 희석. 주 :이 희석 상피 브랜칭을 손상시키지 않고 3D 상태 LG를 유지하는데 최적 인 것으로 밝혀졌다. 그러나, LG의 배양을위한 필수적인 것은 아니다. </li><li> 필터에 또는 라미닌에 5 LG - 4 놓습니다. 문화 LGS 생체 24 - 48 시간 (그림 3) 또는 (20) qPCR에 의한 추가 분석을 위해 분 또는 면역 염색 (그림 4) 4 % PFA로 고정한다. 배아 유선 조직의 qPCR의 분석을 위해, Rebustini 등의 알을 참조하시기 바랍니다. 2011 년. 배아 선의 조직의 면역 형광 분석을 위해 다음의 인용을 참조하십시오 : 호프만 외, (2002)과 스타인버그 전자.t 알., (2005). </li></ol> 3. 마우스 출생과 성인 눈물샘 (LG) : 해부 <ol><li> 적절한 동물 윤리위원회의 기준에 따라 출생 후 마우스를 안락사. 출생 후의 새끼 하루 8 세 이하의 경우, 멸균 가위로 머리를 절단에 의해 안락사. 출생 후 8 일 또는 그 이상을 위해, 70 % 에탄올 모피 스프레이. </li><li> LG를 찾으려면, 마우스 위에서 조명과 해부 현미경 아래에 좌우 측면 누워. 참고 : 산후 성인 LG는 경동맥, 턱 근육과 진피 (그림 5 참조)과 마주하고 있습니다. </li><li> 작은 해부 가위를 사용하여, 옆으로 LG를 배치 할 위치에서 표피에 작은 절개를합니다. </li><li> 집게를 사용하여 LG를 노출 귀쪽으로 표피를 당겨 엽니 다. </li><li> 부드럽게 주변 조직에서 LG를 풀고 집게를 사용합니다. </li><li> LG가 주변 조직에서 분리되면, 조심스럽게, 덕트가 LG를 제거덕트와 눈을 둘러싸는 상피 (도 1)를 연결하는 지점에 의해 파지. </li><li> RT-PCR을위한 RNA 용해 버퍼에 LGS를 배치, 또는 면역 염색에 20 분 동안 4 % PFA로 고정한다. </li></ol>

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저자가 공개하는 게 없다.

문화 다양성과는 LG 전 생체 내 개발 연구를위한 중요한 이점을 제공 조작 할 수 있습니다. 이는 연구자가 가설과, 신경, 및 mesencyhmal 내피 세포 기관을 형성하기 위해 상호 작용하는 방법 상피 평가하기 위해 수행 될 수있는 다수의 섭동을 테스트 할 수있는 속도를 포함한다. 이 모델을 이용할 때 그러나,주의의 수가 존재한다. 첫째, 격리 덕분에 선이 더 이상은 신경이나 혈관 (9, 10)에</su...

누선 (LG)는 시력과 건강 유지 및 안구 표면의 세포의 보호에 중요한 수성 눈물 분비를 담당한다. 안구 자극, 빛 감도 특징과 비전 (1)를 감소 수성 결핍 안구 건조 질환 : 가장 일반적이고 쇠약 안구 질환 중 하나 LG 부전 결과. 인간에서 LG는 눈의 측면 끝 위의 궤도에 상주하는 3 - 안구 표면에 5 배설 덕트 예금 눈물. 마우스가 주요 안구 땀샘이 세 켤레를 가지고,이 대부분의 연구는 하나의 배설 덕트를 통해 눈에 여행 눈물 귀 (exorbital)에 눈물샘 (LG)에 위치한 전방 및 복부입니다. 다른 선의 기관과 마찬가지로, LG는 압축 된 중간 엽 내의 단일 상피 꽃 봉오리가 여러 새싹의 라운드와 용 덕트 형성을 겪게되는 상피 분기 형태 형성의 과정을 통해 개발(그림 1) 분비 샘 꽈리와 덕트의 복잡한 상호 연결된 네트워크를 평방 미터. 개발 과정에서 상피 세포는 혈관된다뿐만 아니라 크게와 우수한 경추 신경절 3에서 교감 신경에 의해 pterygopalatine 신경절의 부교감 신경에 의해 낮은 정도에 분포하는. 유형은 신경 상피, 내피 세포 및 간엽 즉 이들 세포 간의 상호 작용은 각각, 함수 및 성인 조직의 유지에 필수적이다. 그러나, 인터 ​​- 셀 타입의 통신이 이러한 프로세스를 안내하는 방법뿐만 아니라 LG 발달 및 재생을 조정 기본 분자 메커니즘은 확실하지 않다. 배아 체외 배양 기술의 출현은 다수의 분기 장기 4의 발달 및 재생 경로의 식별을 허용했다. 생체 외에서 배양하면 연구자에게 (나 기관을 조작하는 기능을 제공기계적인, 유전 물질 또는 화학 물질) 정의 된 조건에서뿐만 아니라 실시간으로 장기 개발 및 세포 - 세포 상호 작용의 특성을. 마우스의 exorbital LG 전자는이 기술에 매우 의무가 최근 연구 개발 2.5을 조절하는 신호 시스템 정의했습니다. 그러나, LG 개발 및 재생을 뒷받침 분자 단서를 이해 할 필요에도 불구하고 현재 가능성 때문에 장기를 단리에 기술적 인 문제로 남아 파악 하였다. 이 논문에서, 우리는 분리와 개발 프로그램을 정의하기 위해 배아 쥐 LG의 체외 배양을 수행하는 방법에 대해 설명합니다.

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			<td height="21" style="height:21px;width:428px;">Name</td>
			<td style="width:129px;">Catalog #.</td>
			<td style="width:213px;">Company</td>
			<td style="width:997px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 without HEPES</td>
			<td>SH3027101</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>P0781</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin solution from bovine serum</td>
			<td>A9576</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% Solution</td>
			<td>15710</td>
			<td>Electron Microscopy Sciences</td>
			<td>Diluted to 4% in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 10X</td>
			<td>BP665-1</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 1X</td>
			<td></td>
			<td></td>
			<td>Prepared from 10X stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">holo-Transferrin bovine</td>
			<td>T1283</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">L- Ascorbic acid (Vitamin C)</td>
			<td>A4544</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite 100mm Tissue Culture Treated Dishes</td>
			<td>130182</td>
			<td>Thermo Scientific</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Microscope with substage iluminator</td>
			<td>Stemi 2000</td>
			<td>Carl Zeiss</td>
			<td rowspan="2">Any stereo dissecting microscope can be used that has a transmitted light base.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substage Illuminator Base for Stereo Microscope</td>
			<td>TLB 4000</td>
			<td>Diagnostics Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35mm Tissue Culture Treated Dishes</td>
			<td>353001</td>
			<td>Corning</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mm uncoated glass bottom dishes</td>
			<td>P50G-1.5-14-F</td>
			<td>MatTek Corporation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Widefield fluorescence microscope</td>
			<td>Axio Observer Z1</td>
			<td style="width:213px;">Carl Zeiss</td>
			<td>Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached 
			digital camera.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>TCS SP5</td>
			<td style="width:213px;">Leica Microsystems</td>
			<td>Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SWISS Micro-Fine Forceps, #5 (11.2cm)</td>
			<td>17-305X</td>
			<td>Integra LifeSciences Corporation</td>
			<td>Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Standard Tip Forceps, #5 (11cm)</td>
			<td>91150-20</td>
			<td>Fine Science Tools (USA), Inc.</td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reusable Plastic Surgical Knife Handles, style no. 3.</td>
			<td>4-30</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Sterile Surgical Blades, no. 10</td>
			<td>4-310</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous-Micro Total RNA Isolation Kit</td>
			<td>AM1931</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cultrex 3D Culture Matrix Laminin I</td>
			<td>3446-005-01</td>
			<td>Trevigen</td>
			<td>6 mg/ml stock diluted 1:1 with DMEM/F12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Timed-pregnant Crl:CD1(ICR) mice</td>
			<td></td>
			<td style="width:213px;">Charles River Labs</td>
			<td>Embryos are harvested on day 14 (with day of plug discovery designated as day 0).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nucleopore Track-Etched Hydrophilic Membranes, 0.1 um pre size, 13mm</td>
			<td>110405</td>
			<td>Whatman</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Timed-pregnant Pax6-Cre,GFP mice</td>
			<td>Tg(Pax-Cre, GFP)1Pgr</td>
			<td>The Jackson Laboratory</td>
			<td>Optional mice for learning how to locate the LG.</td>
		</tr>
	</tbody>
</table>

manipulating the murine lacrimal gland

누선 (LG)는 각막의 구조 및 기능, 시력에 필수적인 투명한 조직을 유지시키기 위하여 필요한 수성 눈물 분비. 5 관 - 인간의 단일 LG는 3를 통해 안구 표면에 눈물을 전달하는 각각의 눈의 좌우 끝 위의 궤도에 있습니다. 마우스가 주요 안구 땀샘이 세 켤레를 가지고,이 대부분의 연구는 귀에 exorbital 눈물샘 (LG)에 위치한 전방 및 복부입니다. 다른 선의 기관과 마찬가지로, LG는 압축 된 중간 엽 내의 단일 상피 싹이 분비 샘 꽈리와 덕트의 복잡한 상호 연결된 네트워크를 형성 꽃 봉 오리와 덕트 형성의 여러 라운드를 거쳐하는 상피 분기 형태 형성의 과정을 통해 개발하고 있습니다. 이 정교한 과정이 아니라 췌장과 침샘 다른 많은 상피 기관에 설명하고있다. 그러나 LG는 훨씬 더 탐구하고있다 및 형태 형성을 제어하는​​ 메커니즘은 제대로 아래에 있습니다서 있었다. 우리는 모델 시스템으로이 과소 표현이 찾는 해부와 LG 배양과 관련된 어려움의 결과라고 생각한다. 따라서, 우리가 해부 수확 배아 및 산후 LG에 대한 기술과 조직의 체외 배양 방법을 설명합니다.

LG는 상피 분기 형태 형성의 과정을 통해 개발하고 있습니다. E14, E15, E16, E17, 및 P2에서 해부 배아 LGS의 시야 이미지는이 이벤트를 보여줍니다. <img alt="그림 1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> 그림 1. LG는 형태 형성 분기 상피의 과정을 통해 개발하고 있습니다. 갓 E14, E15, E16, E17 및 P2에서 해부 배?...

Watch this Scientific Journal Video about Manipulating the Murine Lacrimal Gland at JoVE.com

Manipulating the Murine Lacrimal Gland

눈물샘 (LG)는 비전과 눈 건강을 유지하기 위해 필요한 눈물의 수성 부품을 생산하는 분기 기관이다. 여기에서 우리는 LG 개발에 참여 경로 신호 해독 쥐 LG 절제술 및 체외 배양 기술에 대해 설명합니다.

쥐의 눈물샘을 조작

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Research

JoVE Journal

Biology

20.7K 조회수.  University of California San Francisco.  눈물샘 (LG)는 비전과 눈 건강을 유지하기 위해 필요한 눈물의 수성 부품을 생산하는 분기 기관이다. 여기에서 우리는 LG 개발에 참여 경로 신호 해독 쥐 LG 절제술 및 체외 배양 기술에 대해 설명합니다. 

비디오: Manipulating the Murine Lacrimal Gland

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

세포 세포 상호 작용을 조작에 대한 실리콘 마이크로 칩

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

연구

생물학

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

체외에서 분리된 뉴런을 조작에 대한 레이저 족집게를 사용하여

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

Murine Renal Transplantation Procedure

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

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

Murine 신장 이식 절차

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

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

마우스 침샘에 줄기 세포의 분리

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

침샘 조직 문화의 유전자 변형과 재결합

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

마우스 유선 화 된 약물 전달을위한 췌관 주입

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

쥐과 밸브 내피 세포의 분리

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

마우스 유선의 냉동 섹션에 간접 면역 형광

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

쥐 관상 동맥 혈관 평활근 세포의 분리

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.