Name: an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks
Uploaded: 2017-02-09T14:00:00.000+00:00
Duration: 6 min 54 s
Description: Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

모든 동물 실험 및 절차는 튤 레인 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 1. 수술 절차 설정 <ol><li> 오토 클레이브 기기, 수술 소모품 및 수술 이전에 문화 용품. 각각의 쥐에 대한 외과 용품은 다음과 같습니다 : 1 드레이프, 사전 절단 구멍 1 드레이프 (0.5 X 1.5) 중앙에, 거즈 패드, 1 흡수 underpad. 수술 도구는 다음과 같습니다 숫자 10 블레이드 1 메스, 핀셋 2 쌍, 미세 가위를. 1 드레이프, 핀셋 1 쌍, 폴리 카보네이트 필터를 6 웰 플레이트 삽입을 준비 : 문화 용품 등이 있습니다. </li><li> 플렉시 글라스 플랫폼 외과 단, 70 % 에탄올 외과 탁상 공간 소독. 멸균 그릇에 사용할 때까지 수술 단계를 유지합니다. <ol><li> 100 밀리미터 배양 접시의 중앙에 구멍에서 1 약 2 드릴링하여 수술 단계를 만듭니다. 다음, 부드럽게 사포를 사용어떤 날카로운 모서리 및 조직에 대한 제기 표면을 생성하기 위해 구멍의 가장자리에 실리콘 접착제의 레이어를 추가합니다. </li><li> 대안 적으로, CAD 소프트웨어를 사용하여 수술 단계를 설계 및 3-D 인쇄 (도 1)로 만든다. </li></ol></li><li> 아래로 멸균 흡수 underpad를 놓고 그 위에 플렉시 글라스 플랫폼을 배치합니다. 흡수성 underpad 옆에 가열 패드에, 미리 잘라 구멍없이 드레이프를 놓습니다. </li><li> 멸균 인산 완충 식염수 (PBS) 사전 따뜻한 매체 및 37 ℃로 식염수. 다음 수술 설치에 50 ML 원뿔 튜브 가열 패드와 장소 식염수 꼭대기 별도의 배양 접시에 장소 미디어와 PBS. </li><li> 모든 패키지는 모든 재료의 멸균 처리를 보장하기 위해 수술의 시작 이전에 개방되어 있는지 확인합니다. 이 절차에 사용되는 일반적인 도구의 전체 목록이 나열되어 특정 외과 재료 및 도구의 표. </li></ol> 2. Mesentery를 조직 수확 <ol><li> 사용 성인 남성의 Wistar 쥐 (350 ± 25g 6 - 생후 8 주). 다른 균주와 쥐의 나이는 대체 할 수 있습니다. </li><li> 케타민 (80 ㎎ / ㎏ 체중)과 자일 라진 (8 ㎎ / ㎏ 체중)의 근육 내 주사를 통해 쥐를 마취. 쥐가 반사 응답을 확인하기 위해 발가락 사이에 집어 마취하에 확인; 아무도이 없어야합니다. 이 단말기 절차에 대한 선제 진통제는 필요하지 않습니다. </li><li> 복부를 면도와 제모 크림을 사용하여 나머지 머리를 제거합니다. 포비돈 - 요오드 다음 70 % 이소 프로필 알코올로 두 번 복부 피부를 닦아주십시오. 와이프를 들어 외과 수술 부위의 중심에서 시작해야하며, 이전에 멸균 거즈 또는 멸균 면봉 동일한 조각으로 세정 된 영역이 겹치지로서 원형 방식으로 제조 된 영역의 외부로 이동한다. 그런 다음 무균 수술 설정에 동물을 전송하고 플렉시 글라스 작은지면 위에 배치형태. </li><li> 흉골 아래에서 일을 시작 장내에서 1.25 절개에 - 메스 블레이드를 사용하여, 0.75에서합니다. 창자 또는 장간막 (피부 1 층, 결합 조직의 1 층 및 근육의 1 층)에 구멍 않도록주의하십시오. </li><li> 절개를 통해 사전 컷 구멍이있는 드레이프를 놓고 드레이프 위에 무균 수술 단계를 배치합니다. 절개와 열 정렬합니다 확인하십시오. 찾아 수술 단계의 개구부를 통해 회장을 끌어 멸균 면봉 어플리케이터를 사용합니다. </li><li> 면봉 어플리케이터를 사용하여 스테이지를 통해 8 장간막 창 및 창 (그림 1)을 만지지 않도록주의 - 6 당깁니다. 조직은 일반적으로 맹장 근처부터 소장 회장 영역으로부터 수확된다. 솔루션을 물방울에 멸균 주사기를 사용하여 필요에 따라 가온 멸균 생리 식염수와 습기에 노출 조직을 유지합니다. </li><li> 펜토 바르 비탈 나트륨의 심장 내 주사를 통해 쥐 (쥐 당 0.2 ml)에 안락사. 저를 제거하기 전에senteric 창, 쥐가 마음을 palpating에 의해 안락사되어 있는지 확인합니다; 맥박이 없어야합니다. </li><li> 창을 절단하는 지방 패드 및 미세 가위를 잡기 위해 핀셋을 사용하여 원하는 장간막 조직을 제거합니다. 창 주위에 지방 (2mm)의 테두리를 남겨주세요. 미디어에 한 번 따뜻하게 멸균 PBS에 한 번 조직을 씻으십시오. </li><li> 복강에 표면화 회장을 돌아 제도적 지침에 따라 동물 폐기하십시오. </li></ol> 타임 랩스 연구 3. 장간막 조직 문화 <ol><li> 멸균 층류 후드로 전송 멸균 문화 용품 (1.1 절 참조) 조직. </li><li> 폴리 카보네이트 필터 멤브레인 위에 각 조직을 전송하는 핀셋을 사용합니다. 지방 패드로 잡아 조직은 혈관의 손상을 방지합니다. </li><li> 빠르게 창을 만지지 않도록 조심하면서, 지방 패드를 사용하여 조직을 확산. 6 웰 플레이트의 바닥에 티슈로 삽입 전환과 미디어의 3 ㎖ (그림 1 커버 </st룽&gt;). 이 절차에 사용되는 일반 용지 1 % 페니실린 스트렙토 마이신 (PenStrep) 및 10 % 소 태아 혈청 (FBS)을 가진 최소 필수 매체 (MEM)를 포함한다. 미디어 혈관 형성을 자극하는 다른 혈청 및 / 또는 성장 인자를 보충 할 수있다. </li><li> 최대 5 일 동안 표준 보육 환경의 각 조직과 문화 (5 % CO 2, 37 ° C) 3.3 - 반복 3.2 단계를 반복합니다. </li></ol> 장간막 조직 4. 타임 랩스 영상 <ol><li> 촬상 당일 공액 BSI 렉틴과 각 웰에 용지를 보충하고, 30 분 동안 표준 배양 조건 하에서 배양한다. 렉틴이없는 미디어로 두 번 조직을 씻으십시오. BSI-렉틴의 얼룩은 문화에서 최대 3 일 동안 장간막 조직에 계속 표시됩니다. </li><li> 현미경 스테이지에 접시를 전송합니다. 자신의 형태와 네트워크 구조를 기반으로 혈액과 림프 혈관을 확인합니다. </li><li> 각 조직에서 원하는 네트워크 영역을 찾아 이미지를 가져 가라. 이미징에주의하십시오같은 지역을 보장하기 위해 위치 이후의 이미지 캡처됩니다. 전동 무대를 사용하는 경우, 좌표를 문서화합니다. </li><li> 인큐베이터에 조직을 돌려 원하는 끝 지점까지 문화를 계속합니다. 반복 4.1 단계 - 4.3 원하는 실험 시점에 따라 필요. </li></ol> 5. 조직 Immunolabeling <ol><li> BSI-렉틴 라벨 <ol><li> 미디어 두 린스 다음 미디어 1시 40분 FITC - 복합 렉틴 (6 웰 플레이트에 웰 당 2.5 mL의 항체 용액) 37 ° C에서 30 분 동안 조직을 품어. 린스를 들어, 미디어를 추가 한 다음 즉시 교체하십시오. </li></ol></li><li> 라이브 / 죽은 라벨링 <ol><li> 1 37 ° C에서 10 분 동안 조직을 품어 : 500 2 MM의 에티 디움 호모 다이머-1 및 1 : 미디어 두 린스 다음 미디어 500 1 ㎜의 칼 세인의 AM (6 웰 플레이트에 웰 당 2.5 mL의 항체 용액). </li></ol></li><li> BSI-렉틴 / NG2 라벨 <ol><li> 현미경에 확산 조직 하락전자 (1-2 조직 / 슬라이드) 및 건조 할 수 있습니다. 지방을 절제하기 위해 단단히 눌러 메스와 여분의 지방을 제거합니다. </li><li> -20 ° C에서 30 분 동안 냉 메탄올에서 조직을 고정한다. PBS (3 × 10 분)으로 조직을 씻으십시오. </li><li> 100 토끼 폴리 클로 날 항체 NG2 5 % 정상 염소 혈청 (NGS) 일차 항체를 라벨링 한 실온에서 1 시간 동안 조직을 배양한다. PBS (3 × 10 분)으로 조직을 씻으십시오. </li><li> 이차 항체를 라벨링 한 실온에서 1 시간 동안 조직 배양한다 : 100 염소 항 - 토끼 항체 CY2 공역 (GAR-CY2) 5 %를 NGS. PBS (3 × 10 분)으로 조직을 씻으십시오. </li><li> PBS로 두 린스 다음 PBS에서 1시 40분 FITC - 복합 렉틴과 실온에서 30 분 동안 조직을 품어. 린스를 들어, PBS를 추가 한 다음 즉시 교체하십시오. </li><li> 슬라이드를 마운트하려면, 위에 50:50 PBS와 조직과 글리세롤 솔루션 및 장소 coverslip에 커버. 매니큐어를 사용하여 슬라이드 가장자리를 밀봉합니다. </li></ol></li><li> LYVE-1 / PECAM 라벨<ol><li> 현미경 슬라이드에 조직을 확산 (1-2 조직 / 슬라이드) 및 건조 할 수 있습니다. 지방을 절제하기 위해 단단히 눌러 메스와 여분의 지방을 제거합니다. </li><li> -20 ° C에서 30 분 동안 냉 메탄올에서 조직을 고정한다. PBS + 0.1 % 사포닌 (3 × 10 분)와 조직을 씻으십시오. </li><li> 200 마우스 모노클 비오틴 CD31 항체와 1 : 일차 항체를 라벨링 한 실온에서 1 시간 동안 조직 배양한다 (100) 토끼 폴리 클로 날 LYVE-1 항체를 PBS + 0.1 % 사포닌 + 2 % 소 혈청 알부민 (BSA) + 5 % NGS . PBS + 0.1 % 사포닌 (3 × 10 분)와 조직을 씻으십시오. </li><li> 500 CY3 접합 스트렙 타비 딘 항체 및 1 : PBS + 0.1 % 사포닌 + 2 % BSA + 5 % NGS 100-GAR CY2 차 항체의 표지의 경우, 1 일 실온에서 시간 동안 조직을 배양한다. PBS + 0.1 % 사포닌 (3 × 10 분)와 조직을 씻으십시오. </li><li> 50:50 PBS와 글리세롤 솔루션 슬라이드, 커버 조직을 탑재하고 상단에 커버 슬립을 배치합니다. 매니큐어를 사용하여 슬라이드 가장자리를 밀봉합니다. </li></ol></li><li> BrdU의 / BSI-렉틴 라벨링 <ol><li> 미디어에 1 ㎎ / ㎖ BrdU의 추가 및 BrdU의 솔루션으로 조직 미디어를 교체합니다. 37 ° C에서 2 시간 동안 품어. </li><li> 현미경 슬라이드에 조직을 확산 (1-2 조직 / 슬라이드) 및 건조 할 수 있습니다. 지방을 절제하기 위해 단단히 눌러 메스와 여분의 지방을 제거합니다. </li><li> -20 ° C에서 30 분 동안 냉 메탄올에서 조직을 고정한다. PBS (3 × 10 분)으로 조직을 씻으십시오. </li><li> 37 ° C에서 1 시간 동안 2 M 염산으로 조직 DNA 변성. PBS + 0.1 % 사포닌 (3 × 10 분)에 조직을 씻으십시오. </li><li> PBS + 0.1 % 사포닌 + 2 % BSA + 5 % NGS 100 모노클로 날 마우스 항 BrdU의 : 차 항체의 표지의 경우, (1)과 실온에서 1 시간 동안 조직을 배양한다. PBS + 0.1 % 사포닌 (3 × 10 분)와 조직을 씻으십시오. </li><li> 이차 항체 라벨링 1 실온에서 1 시간 동안 조직 배양한다 : 100 염소 항 - 마우스 Cy3가 접합 된 항체를 PBS + 0.1 % 사포닌 + 2 % BSA + 5 % NGS의 (GAM-Cy3에 참조). PBS + 0.1 % 사포닌 (3 × 10 분)와 조직을 씻으십시오. </li><l난&gt; PBS로 두 린스 다음 PBS에서 1시 40분 FITC - 복합 렉틴과 실온에서 30 분 동안 조직을 품어. </li><li> 상단에 슬라이드, 커버 조직 50:50 PBS와 글리세롤 솔루션 및 장소 coverslip에 마운트합니다. 매니큐어를 사용하여 슬라이드 가장자리를 밀봉합니다. </li></ol></li><li> BSI-렉틴 / CD11b를 라벨링 <ol><li> 현미경 슬라이드에 조직을 확산 (1-2 조직 / 슬라이드) 및 건조 할 수 있습니다. 지방을 절제하기 위해 단단히 눌러 메스와 여분의 지방을 제거합니다. </li><li> -20 ° C에서 30 분 동안 냉 메탄올에서 조직을 고정한다. PBS + 0.1 % 사포닌 (3 × 10 분)와 조직을 씻으십시오. </li><li> 일차 항체를 라벨링 한 실온에서 1 시간 동안 조직 배양한다 : PBS + 0.1 % 사포닌 + 2 % BSA + 5 % NGS 100 마우스 항 래트는 CD11b. PBS + 0.1 % 사포닌 (3 × 10 분)와 조직을 씻으십시오. </li><li> 이차 항체를 라벨링 한 실온에서 1 시간 동안 조직 배양한다 : PBS + 0.1 % 사포닌 + 2 % BSA + 5 % NGS 100 GAM-Cy3에. PBS로 조직을 씻으십시오+ 0.1 % 사포닌 (3 × 10 분). </li><li> PBS로 두 린스 다음 PBS에서 1시 40분 FITC - 복합 렉틴과 실온에서 30 분 동안 조직을 품어. </li><li> 상단에 슬라이드, 커버 조직 50:50 PBS와 글리세롤 솔루션 및 장소 coverslip에 마운트합니다. 매니큐어를 사용하여 슬라이드 가장자리를 밀봉합니다. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

이 프로토콜은 미세 혈관 망 성장 저속 촬상 용 생체 도구 래트 장간막 배양 모델을 사용하는 방법을 설명합니다. 우리 실험실에서 이전 작업은 1 우리의 모델) 혈관 신생 (8), 2) 림프관 8, 3) 주연 세포 - 내피 세포 상호 작용 8, 4) 항 혈관 약물 검사 (9)의 사용을 설립했다. 여러 시점에서 배양 된 쥐 장간막 조직을 이미징 ...

미세 혈관 망의 성장 및 리모델링 조직 기능의 공통 분모, 상처 치유, 및 여러 병리이며 주요 공정은 기존의 1, 2에서 새로운 혈관의 성장으로 정의 혈관이다. 새로운 혈관을 엔지니어링 또는 혈관 신생에 관여하는 세포 역학의 중요성을 이해 혈관 기초 요법을 설계 조직 중요하다. 그러나이 프로세스는 복잡하다. 이것은 미세 혈관 망의 특정 위치에서 변화 여러 종류의 세포 (즉, 내피 세포, 평활근 세포, 혈관 주위 세포, 대식 세포, 줄기 세포) 및 여러 시스템 (림프 네트워크, 신경 네트워크)을 포함 할 수있다. 시험관 내 혈관 신생 모델 (3)에 관련된 다른 셀들 간의 관계를 검사에 엄청난 공헌하지만, 생리 학적 관련성 파묻혀 인해 훼손 될 수있다<html> 제한된 복잡하고 밀접 생체 시나리오를 반영하지 않는다는 사실을 r에. 이러한 한계의 3 차원 배양 시스템을 극복하기 위해 3, 생체 조직의 모델 (4), 마이크로 유체 시스템의 5, 6, 최근 개발되어 소개되었다 7 계산 모델. 그러나, 미세 혈관 손상 네트워크 생체의 혈관 신생을 조사하는 저속 기능이 모델에 대한 필요성이 여전히 존재한다. 복잡성의 수준과 혈관 신생 연구를위한 새로운 시간 경과 모델의 설립은 혈관 신생을 조절하는 기본 메커니즘을 이해하는 귀중한 도구를 제공하고 치료법을 개선 할 수 있습니다. 손상되지 않은 미세 혈관 네트워크를 통해 혈관 신생의 생체 조사를 가능하게하는 잠재적 인 모델은 쥐 장간막 문화 모델&gt; 8. 최근 연구에서, 우리는 혈액과 림프 미세 혈관 네트워크는 문화 후 가능한 남아 있음을 증명하고있다. 더 중요한 것은, 래트 장간막 배양 모델은 기능적 주연 세포 - 내피 세포 상호 작용, 혈액 및 림프관 내피 세포 연결, 저속 촬상을 조사하는데 사용될 수있다. 본 연구의 목적은 시간 경과 영상 법을위한 우리의 프로토콜을 제공하는 것이다. 우리의 대표적인 결과는 혈청과 혈관의 자극 후 생존을 유지하고 조직 특이 적 혈관 반응뿐만 아니라 내피 세포 추적 연구를 정량화이 방법을 사용 예제를 제공하는 여러 세포 유형을 문서화합니다.

<table><tbody><tr><td>Drape</td><td>Cardinal Health</td><td>4012</td><td>12&amp;rdquo; x 12&amp;rdquo; Bio-Shield Regular Sterilization Wraps</td></tr><tr><td>Scalpel Handle</td><td>Roboz Surgical Instrument</td><td>RS-9843</td><td>Scalpel Handle, #3; Solid; 4&quot; Length</td></tr><tr><td>Sterile Surgical Blade</td><td>Cincinnati Surgical</td><td>0110</td><td>Stainless Steel; Size 10</td></tr><tr><td>Culture Dish (60 mm)</td><td>Thermo Scientific</td><td>130181</td><td>10/Sleeve</td></tr><tr><td>Graefe Forcep (curved tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5135</td><td>Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Graefe Forcep (straight tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5130</td><td>Micro Dissecting Forceps; Serrated, Straight; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Noyes Micro Scissor</td><td>Roboz Surgical Instrument</td><td>RS-5677</td><td>Noyes Micro Dissecting Spring Scissors;&lt;br/&gt; Straight, Sharp-Blunt Points; 13 mm Cutting Edge; 0.25 mm Tip Width, 4 1/2&quot; Overall Length</td></tr><tr><td>Gauze Pads</td><td>FisherBrand</td><td>13-761-52</td><td>Non-Sterile Cotton Gauze Sponges; 4&quot; x 4&quot; 12-Ply</td></tr><tr><td>Cotton-Tippled Applicators</td><td>FisherBrand</td><td>23-400-124</td><td>6&quot; Length; Wooden Shaft; Single Use Only</td></tr><tr><td>6-Well Plate</td><td>Fisher Scientific</td><td>08-772-49</td><td>Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td></tr><tr><td>Sterile Syring 5 mL</td><td>Fisher Scientific</td><td>14-829-45</td><td>Luer-Lok Tip</td></tr><tr><td>Sterile Bowl</td><td>Medical Action Industries Inc.</td><td>01232</td><td>32 oz. Peel Pouch; Blue; Sterile Single Use</td></tr><tr><td>6-Well Plate Inserts (CellCrown Inserts)</td><td>SIGMA</td><td>Z681792-3EA</td><td>6-Well Plate Inserts; Non-Sterile</td></tr><tr><td>Polycarbonate Filter Membrane</td><td>SIGMA</td><td>TMTP04700</td><td>Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, White Plain</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Beuthanasia</td><td>Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td><td>MWI #: 011168</td><td>Active Ingredient: Per 100 mL, 390 mg pentobarbital sodium, 50 mg phenytoin sodium&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Kateset 100 mg/mL</td></tr><tr><td>Xylazine</td><td>LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Anased 100 mg/mL</td></tr><tr><td>Saline</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>MEM</td><td>Invitrogen</td><td>11095080</td><td></td></tr><tr><td>PenStrep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>BSA</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>SIGMA</td><td>S7900-100G</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>Fisher Scientific</td><td>S25372</td><td></td></tr><tr><td>Povidone-Iodine</td><td>Operand</td><td>82-226</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>SIGMA</td><td>320331</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>FITC-conjugated Lectin</td><td>SIGMA</td><td>L9381-2MG</td><td></td></tr><tr><td>Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td><td>SIGMA</td><td>AB5320</td><td></td></tr><tr><td>PECAM (CD31) Antibody</td><td>BD Biosciences</td><td>555026</td><td></td></tr><tr><td>LYVE-1 Antibody</td><td>AngioBio Co.</td><td>11-034</td><td></td></tr><tr><td>Goat Anti-Rabbit Cy2-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>111-585-144</td><td></td></tr><tr><td>Goat Anti-Mouse Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>115-227-003</td><td></td></tr><tr><td>Streptavidin Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>016-160-084</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>5-Bromo-2&#039;-Deoxyuridine</td><td>SIGMA</td><td>B5002</td><td></td></tr><tr><td>Monoclonal Mouse Anti-Bromodeoxyuridine&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Clone Bu20a</td><td>Dako</td><td>M074401-8</td><td></td></tr><tr><td>Mouse Anti-Rat CD11b&amp;nbsp;</td><td>AbD Serotec</td><td>MCA275R</td><td></td></tr></tbody></table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

혈관 신생은 기존의 혈관으로부터 새로운 혈관의 성장과 같이 정의, 내피 세포, 혈관 주위 세포, 평활근 세포, 면역 세포 및 림프관 및 신경과 조정을 포함한다. 멀티 셀, 멀티 시스템의 상호 작용은 생리 학적으로 관련 환경에서 혈관 신생의 조사를 필요로한다. 시험관 내 세포 배양 모델의 사용은 기계적인 통계를 제공 한 상태 즉, 일반적인 비판은 미세 혈관 네트워크와 연관된 복잡성 요점을 되풀이하지 않는다는 것이다. 이 프로토콜의 목적은 전 배양 된 쥐 장간막 조직에서 혈관 신생을 자극 한 후 그대로 미세 혈관 망의 경과 비교를 할 수있는 능력을 입증하는 것이다. 배양 된 조직은 계층 구조를 유지하는 미세 혈관 네트워크를 포함한다. 면역 표지 내피 세포, 평활근 세포, 혈관 주위 세포, 혈관과 림프관의 존재를 확인한다. 안에ddition는 BSI-렉틴과 조직을 라벨은 이전과 증가 된 모세 혈관의 발아 및 혈관 밀도를 특징으로 혈청 성장 인자의 자극 후 로컬 네트워크 영역의 시간 경과 비교를 할 수 있습니다. 일반적인 세포 배양 모델에 비해,이 방법은 생리 학적으로 관련 미세 혈관 내피 세포 네트워크의 계통 연구 및 조직 특이 적 혈관 형성 약물 평가하는 툴을 제공한다.

문화 3 일 후, 조직은 쥐 장간막 문화 모델 (그림 2A)의 미세 혈관의 가능성을 입증하는 라이브 / 죽은 생존 / 세포 독성 키트와 함께 표시 하였다. 장간막에 존재하는 세포의 대부분은 미세 혈관 내피 세포에서 세그먼트의 위치에 기초하여 식별 된 배양 가능한 남았다. 내피 세포의 증식은 렉틴 / BrdU의 라벨링 (도 2D)에 의하여 확인 하였다. 따라서 ?...

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An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

<em&gt; 전의 VIVO</em&gt; 방법 배양 된 쥐 장간막 미세 혈관 네트워크의 타임 랩스 영상에 대한

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

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Developmental Biology

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

6.8K 조회수.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

비디오: An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

생체 외 및 생체 내 모델에서 혈관 벽에서 흐름 조건에 세균 부착을 연구하기 위해

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

연구

면역학 및 감염병학

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

실시간으로 마취 마우스에 생체 내에 현미경으로 장간막 혈관에서 호중구와 단핵구 이미징

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

쥐 장간막의 Angiogenesis 분석

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

생물학

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

쥐의 장간막의 Exteriorization : Angiogenesis에 관련된 세포 역학을 조사하기위한 모델

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

쥐의 고립 된 저항 동맥의 비디오 현미경을 이용 하 여 혈관 제어 메커니즘의 평가

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

의학

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

에 대한 마우스 태아의 전체 소장 문화 시스템<em&gt; 전의 VIVO</em&gt; 신호 전달 경로와 융모 개발의 3 차원 라이브 영상의 조작

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

마우스 심장의 모세혈관, 동맥 및 페리사이클의 동적 측정 및 이미징

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

세포 내 칼슘의 두 광자 이미징<sup&gt; 2 +</sup&gt; 고립 된 쥐 대동맥의 취급 및 내피 세포에서 산화 질소 생산과 부드러운 근육 세포

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusion 기술은 쥐 장간막에서 미세 혈관 투과성의 규정을 조사하기 위해

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

개발 및 특성화의<em&gt; 체외</em&gt; 미세 혈관 네트워크 및 내피의 정량적 측정 [칼슘<sup&gt; 2 +</sup&gt;]<sub&gt; 전</sub&gt; 및 질소 산화물 생산

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

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