시약 준비 <ol><li> BSA (0.5 %), 2 MM EDTA (에틸렌 다이아 민 테트라 초산)과 PBS를 포함하는 버퍼를 확인합니다. 공기 거품이 맥 분리 열을 차단할 수 있기 때문에, 버퍼는 2-8 ° C에서 사용하기 전에 degassed하고 저장해야합니다. </li><li> 우리는 5 % 마우스 혈청을 포함하는 RPMI 1640 배지를 사용합니다. 문화 매체는 이러한 화합물은 세포 자극의 특이성을 변경하므로, BSA 또는 FCS를 포함해서는 안됩니다. </li><li> 마우스 IL - 17 분비 분석 - Miltenyi Biotec에서 셀 - 농축 및 탐지 키트. IL - 17 캐치 시약, IL - 17 탐지 항체 (비오틴), 안티 - 비오틴 - PE 및 안티 체육 MicroBeads : 키트는 다음과 같은 구성 요소가 포함되어 있습니다. </li></ol> Splenocytes을 자극 <ol><li> 이 프로토콜은 이러한 unstimulated splenocytes 및 T 세포에 대한 counterstain로 부정과 긍정적인 컨트롤의 존재에서 수행됩니다. 이 프로토콜은 살균 기술을 사용하여 수행됩니다. </li><li> gentleMACS ™의 Dissociator를 사용하여 격리되었다 마우스 splenocytes의 단일 세포 현탁액을 준비합니다. 세포의 농도는 세포 계수를 통해 미리 정해진해야합니다. 실온에서 10 분간 200 × g에서 펠렛은 세포. </li><li> 원심 분리 다음, 피펫을 사용하여 펠렛에서 뜨는을 대기음. 펠렛의 손실을 피하기 위해 튜브를 가만히 따르다하지 마십시오. </li><li> 이제 문화 매체에있는 세포를 resuspend 다음 잘 추가할 수 있습니다. ML 당 천만 세포 cm2 당 오백만 세포의 농도에 대한 충분한 매체를 추가합니다. </li><li> 우리 resuspended 세포에 면역 반응을 촉진하기 위해, 우리는 샘플을 ionomycin (1 μg / ML)와 PMA (10 NG / ML) 추가하고 부드럽게 최대 pipetting 아래로 혼합 솔루션을. 그러면 우물이 적절하게 표시됩니다. </li><li> 이제, 우리는 37 3 시간 동안 우리의 세포를 품어됩니다 ° C 자극 기간을 시작없이 믹싱과 함께. 자극의 시작에서 IL - 17 분석 3 시간 진행되므로 적절하게 계획이다. </li><li> 분비를 중지하려면, 전지가 얼음에 위치하고 부드럽게 감기 버퍼와 아래 pipetting하여 자극을 세포를 수집하고 있습니다. 세포는 다음 우물에서 튜브로 전송하고 두 번째 시간을 씻어 있습니다. </li><li> 모든 세포가 수집되는 것을 보장하기 위해, 그것은 현미경 아래에 접시를 확인하는 것이 좋습니다. 세포가 여전히 남아 첨부하면 감기 버퍼와 접시를 rinsing하여 남아있는 세포를 수집할 수 있습니다. 당신의 세포 현탁액의 모든 세포 대단히 짧은 시간은 미리 분리 필터를 사용하여 제거할 수 있습니다. </li></ol> 캐치 시약으로 세포를 라벨링 <ol><li> 그것은 IL - 17 - secreting 세포의 2 % 미만이있는 경우에는이 분석은 최적의 작동, 참고 파라마운트입니다. </li><li> IL - 17 secreting 세포의 농도가 적절하게 볼륨을 조절할 이상 2퍼센트 것으로 예상하는 경우. <img src="/files/ftp_upload/old/1037_2008_12_8_6/figure8.png" alt="그림 8" width="500" height="162" /></li><li> 이 절차의 함정 하나 가능성 BI 특정 항체가되므로 잘못된 반응을 생성, 비 secreting의 T 세포와 인접 림프구에서 분비 트랩 IL - 17에 바인딩 할 때 레이블링하는 동안 발생할 수있는 캐치 시약의 교차 오염이다. </li><li> 이 문제를 회피하기 위해서는 IL - 17의 보급을 저하이 교차 오염을 피하기 위해 아래 사전 추위 버퍼와 라벨 부착 및 작동하도록 냉각 세포에 중요합니다. 세포 차가운 유지 이외에, 그들은 정의 농도에 보관해야합니다. </li><li> 라벨 절차를 시작하기 위해, 우리는 15 ML closable 튜브 천만 세포를 사용합니다. 높은 세포 숫자가 사용할 수있다면, 간단하게 따라 모든 볼륨을 최대 규모. 최적의 세포 농도를 얻을되면 추위 버퍼 10 ML을 추가하여 세포를 씻으십시오. </li><li> 냉장 원심 분리기 10 분 (2-8 ° C) 300에서 전지 × g를 스핀 다운. </li><li> 원심 분리에 따라 완전히 피펫을 사용하여 표면에 뜨는를 대기음. 이 세포 손실 및 이란게 부정 확한 볼륨을 이끌로 뜨는을 가만히 따르다하지 마십시오. 감기 버퍼, 원심 분리하고, 열망의 10ml를 추가로 구성되어 세척 단계를 반복합니다. </li><li> 이제 원하는 순도의 펠렛을 가지고, 차가운 문화 매체 80 μL의 세포를 resuspend. 그들을 분류하기 위해, 우리는 이제 마우스 IL - 17 캐치 시약 20 μL를 추가합니다. </li><li> 얼음 5 분 동안 세포를 품어. </li><li> 얼음에 5 분 잠복기 후, 튜브를 제거하고 37 ° C 따뜻한 매체 10 ML에있는 세포를 희석. 다음 MACSmix 튜브의 회전에 튜브를 확보하고 37 튜브를 품어 ° C를 지속적으로 움직임에 따라 45 분. 37 온도를 증가하는 것은 ° C는 시토킨 분비를 다시 시작합니다. </li></ol> IL - 17 탐지 항체 (비오틴) 및 안티 - 비오틴 - PE와 세포를 라벨링 <ol><li> 37 ° C에서 45 분 분비 기간 후, 얼음에 즉시 튜브를 놓으십시오. 이것은 시토킨 분비를 중지합니다. 여기에서 그것에 얼음에 세포를 유지하는 것이 중요합니다.감기 버퍼로 작업하면 캐치 시약의 교차 오염을 방지합니다. </li><li> 2-8에서 300xg에서 추위 버퍼와 원심 분리기 ° 10 분 C로 튜브를 입력합니다. 완전히 뜨는을 대기음. 세척이 한 번 더 단계를 반복합니다. </li><li> 세포 펠렛은 추위 버퍼의 80 μL에 resuspended이며, 튜브는 얼음에 위치합니다. </li><li> 항체의 unspecific 바인딩을 방지하기 위해, 우리는 5 분 얼음에 10μl의 FcR 차단 시약과 부화를 추가하는 것이 좋습니다. 그럼 우리가 얼음에 10 분간 비오틴과 부화 복합이다 마우스 IL - 17 탐지 항체 (비오틴) 20 μL를 추가합니다. </li><li> 다시 한번, 우리는 2-8에서 300xg에서 추위 버퍼와 원심 분리기 10 ML 추가 ° C, 10 분에 의해 전에했던 것처럼 세포를 씻으십시오. </li><li> 지금 뜨는을 대기음 차가운 버퍼 80 μl의 세포 펠렛을 resuspend. </li><li> 우리 이차 항체, 안티 - 비오틴 - PE 20 μL를 추가합니다. </li><li> 세포와 보조 항체 솔루션을 섞어서 다시 얼음에 10 분 동안 튜브를 품어. </li><li> 이 두 번째 배양이 완료되면, 10 ML 감기 버퍼와 원심 분리기로 세포를 씻어. </li><li> 펠렛은 이제 추운 버퍼 500 μL에 resuspended 수 있습니다. </li><li> 세포가 더 하류 분석을 위해 분리 수있다면, 그들은 자기 (磁 气) 안티 - PE - MicroBeads으로 표시하고 Mac에서 열 및 Mac에서 구분 기호를 사용하여 수동 또는 자동 분리 수 있습니다. </li><li> 우리는 이제 splenocytes의 라벨링을 완료 및 분석을위한 준비가되어있다. </li></ol> 외과 분석 <ol><li> 우리의 분석을 위해, 우리는 유동세포계측법에 의해 자극과 unstimulated splenocytes을 비교합니다. </li><li> 흐름 cytometric 분석하기 전에 전지가 죽은 세포 밖으로 게이트 0.5 μg / ML에서 propidium 요오드화물 물들일 수 있습니다. MACSQuant ™ 분석기는 각 샘플에 대한 200,000 개 셀 탑재되어 이제 샘플의 상대적인 항체 반응의 스캐터 플롯을 볼 줄 수 있습니다. 희귀 세포의 분석을 위해 두 가지 중요한 고려 사항 - IL - 17 양성 세포와 같은 - 유동세포계측법에 있습니다 : <ul><li> 앞으로 분산 비교 측면 스캐터 플롯에 lymphocytes에 게이트를 설정 </li><li> 그리고 죽은 세포 (propidium 요오드화물의 물들일)와 B - 세포 (unspecific 배경 얼룩의 원인이 될 수있는)가 게이팅 추가 검출의 감도를 향상시키기 위해 </li></ul></li><li> 우리는 T 세포와 B 세포를 감지하는 CD45R/B220-PerCP 항체를 감지하는 CD4 - APC 항체를 사용했습니다. 우리는 샘플의 전방 및 측면 산란 특성을 확인하고 림프구 인구에 게이트를 적용합니다. </li><li> 두 번째 게이트는 스테인드 죽은 세포와 스테인드 B 세포 (Y 축)를 참조하기 위해 적용되었습니다. 이러한 세포는 T 세포 분석에서 제외됩니다. </li><li> 이 예제에서, 어떤은 본질적으로 우리의 부정적인 제어이며, 우리는 unstimulated 샘플에서 거의 IL - 17 secreting의 CD4 양성 T 세포가있다는 것을 알 수 있습니다 - 약 0.003 % (그림 4). 자극 T 세포 인구를 살펴보면, 우리는 CD4의 상당수 긍정적인 T 세포를 볼 수 분비 IL - 17 - 분리하기 전에 0.367 % 정도 (그림 5A)와 Mac에서 기술 (그림 5B)와 자성 분리 후 60.76 %. </li></ol>

모든 프로토콜 및 데이터 시트 www.miltenyibiotec.com에서 구할 수 있습니다. 경고 시약은 나트륨 azide가 포함되어 있습니다. 산성 조건 하에서 나트륨 azide은 매우 독성 hydrazoic 산을 산출. Azide 화합물은 폐기 전에 물을를 실행에 희석해야합니다. 이러한주의 사항은 폭발 조건이 개발 있습니다 배관 예금을 피하기 위해 권장합니다.

TH17 세포는 적응 면역과 염증 반응에 중요한 역할을하고 면역 염증을 유발하는 측면에서 핵심적인 구성 요소입니다. 이 동영상 Miltenyi의 마우스를 사용하는 방법 문서를 IL - 17 분비 분석 - 세포 강화 및 탐지 키트 (PE). 이 절차를 수행할 때 그것은 당신의 시작 셀 준비 IL - 17 - secreting 세포의 빈도의 추정을하는 것이 중요합니다. 라벨 및 시토킨 분비 기간 동안 적절한 세포 농도가 중단의 다른 세포에 의해 교차 오염을 방지하고 안정적​​인 결과를 보장합니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Mouse IL-17 Secretion Assay Cell Enrichment and Detection Kit (PE) </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-094-213</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx">http://www.miltenyibiotec.com/en/PG_98_1005_Mouse_IL_17_Secretion_Assay_Cell_Enrichment_and_Detection_Kit.aspx</a>
</td>
</tr>
<tr>
<td>RPMI 1640</td>
<td>Medium</td>
<td>Miltenyi Biotec</td>
<td>130-091-440</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx">http://www.miltenyibiotec.com/en/PG_243_293_RPMI_1640.aspx</a></td>
</tr>
<tr>
<td>CD4-APC</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-611</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx">http://www.miltenyibiotec.com/en/PG_599_327_CD4.aspx</a></td>
</tr>
<tr>
<td>CD8a-FITC</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-091-605</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx">http://www.miltenyibiotec.com/en/PG_599_328_CD8a.aspx</a></td>
</tr>
<tr>
<td>Propidium Iodide Solution</td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-093-233</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx">http://www.miltenyibiotec.com/en/PG_337_744_Propidium_Iodide_Solution.aspx</a></td>
</tr>
<tr>
<td>FcR Blocking reagent, mouse </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-092-575</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx">http://www.miltenyibiotec.com/en/PG_601_435_FcR_Blocking_Reagent_mouse_.aspx</a></td>
</tr>
<tr>
<td>Dead Cell Removal Kit </td>
<td>Reagent</td>
<td>Miltenyi Biotec</td>
<td>130-090-101</td>
<td>&nbsp;</td>
<tr>
<td>MACSmix Tube Rotator</td>
<td>Instrument</td>
<td>Miltenyi Biotec</td>
<td>130-090-753</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx">http://www.miltenyibiotec.com/en/PG_34_251_MACSmix_Tube_Rotator.aspx</a></td>
</tr>
<tr>
<td>gentleMACS&#x2122; Dissociator</td>
<td>Instrument</td>
<td>Miltenyi Biotec</td>
<td>130-093-235</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx">http://www.miltenyibiotec.com/en/PG_709_763_gentleMACS_Dissociator.aspx</a></td>
</tr>
<tr>
<td>autoMACS Pro Starting Kit</td>
<td>Instrument</td>
<td>Miltenyi Biotec </td>
<td>130-092-545</td>
<td><a href="http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx">http://www.miltenyibiotec.com/en/PG_651_679_autoMACS_Pro_Starting_Kit.aspx</a></td>
</tr>
<tr>
<td>MACS Columns</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td><a href="http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx">http://www.miltenyibiotec.com/en/NN_115_Columns_at_a_glance.aspx</a></td>
</tr>
<tr>
<td>MACS Separator</td>
<td>Tool</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

detection and isolation of viable mouse il-17-secreting t cells

맥스 시토킨 분비 분석 기술은 단일 세포 수준과 가능한 시토킨 - secreting 세포의 민감한 고립에서 분비 크린 시토킨의 검색이 가능합니다. IL - 17 - secreting 세포, 마우스 splenocytes의 단일 세포 현탁액 ° PMA / ionomycin와 C는 시토킨 분비를 유도하기 위해 37 준비와 자극입니다 레이블하기 위해. 분비 세포를 중지하기 위해서는 그것이 분비와 세포 표면 근처에 잡힌로 leukocytes의 세포 표면과 IL - 17 CD45에 바인딩 이중 특정 항체를 다음 얼음에 게재되고 IL - 17 캐치 시약에 노출되어 있습니다. 분비 그러면 37의 온도를 증가시켜 다시 시작 ° C와 IL - 17은 캐치 시약에 의해 갇혀있다. 분비 그런 다음 얼음에 세포를 배치하여 다시 중지됩니다. 함정 IL - 17을 감지하기 위해 세포는 비오틴에 복합 두번째 IL - 17 특정 항체 및 안티 - 비오틴 - PE 항체와 incubated 있습니다. 셀 이제 직접 유동세포계측법에 의해 분석이나 안티 - PE 복합 MicroBeads과 이후의 라벨링에 의해 분리 및 농축을위한 준비를하실 수 있습니다.

Watch this Scientific Journal Video about Detection and Isolation of Viable Mouse IL-17-Secreting T Cells at JoVE.com

Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

이 절차를 자극시 IL - 17 분비 적극적으로 마우스 TH17의 leukocytes의 탐지 및 격리에 대해 설명합니다.

가능한 마우스 IL - 17 - Secreting의 T 세포 탐지 및 격리

The MACS Cytokine Secretion Assay technology allows detection of secreted cytokines on the single cell level and sensitive isolation of viable ...

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detection-and-isolation-of-viable-mouse-il-17-secreting-t-cells

Research

JoVE Journal

Biology

11.1K 조회수.  Miltenyi Biotec,GmbH.  ChineseVersion JapaneseVersion 

비디오: Detection and Isolation of Viable Mouse IL-17-Secreting T Cells

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

CD4의 분리 + Miltenyi 맥을 정제를 사용하여 마우스 림프절에서 T 세포

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

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Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

절연 및 마우스 배아 새싹 세포의 유도

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

판막병 내피 세포의 분리

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

기본 마우스 Trophoblast 셀 및 Trophoblast의 침략 분석의 절연

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases 2. The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands 3. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways 3.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.

Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.

마우스 기관에서 자연적으로 흐르는 것과 셀 및 Submucosal 글 랜드 덕트 세포의 분리

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

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 ...

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

분리 및 마우스 대동맥 내피 세포의 차 문화

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed&#160;and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

마우스 관상 내피 세포의 분리

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.