This work was funded by start-up funds provided to Dr. Silviya Zustiak by Saint Louis University as well as by a President&#8217;s Research Fund (PRF) grant awarded to Dr. Silviya Zustiak by Saint Louis University. We thank Naveed Ahmed and Keval Shah for technical assistance.

하이드로 겔 관련 솔루션 및 분취 1. 준비 <ol><li> 폴리 아크릴 아미드 겔 전구체 용액의 제조. <ol><li> 아크릴 아미드 (A)을 혼합하여 폴리 아크릴 아미드 겔 전구체 용액을 준비한다 (V / w 40 %, M은 r에 71.08 g / ㏖), 가교제 비스 아크릴 아미드 (B) (V / w 2 %, M은 154.17 g / mol 인 R), 및 탈 표 1에 규정 된 부피 백분율에서 이온수. 참고 :이 솔루션은 큰 일괄 적으로 준비하고 몇 개월까지 4 ° C에 저장할 수 있습니다. <ol><li> 주의 : 아크릴 아마이드는 흡입 또는 섭취시 독성, 특히 분말 형태로 할 때 : 따라서, 바람직하게는 독성의 위험을 줄이기 위해 / V 솔루션 w 40 %를 사용합니다. 이러한 장갑, 고글, 및 실험실 코트로, 보호 옷을 입고있는 동안 만 처리합니다. 냉장고에 차광, 밀폐 된 용기에 보관할 것 (&lt;23 ° C, 영역을 통풍이 잘되는). </li><li> 주의 : 비스 - 아크릴 아미드, 특히, 흡입하거나 섭취시 유독분말 형 : 따라서, 바람직하게는 독성의 위험을 줄이기 위해 / V 솔루션 w 2 %를 사용합니다. 이러한 장갑, 고글, 및 실험실 코트로, 보호 옷을 입고있는 동안 만 처리합니다. 냉장고 (&lt;4 ° C에서, 환기가 잘되는 장소)에서 차광, 밀폐 된 용기에 보관할 것. </li></ol></li></ol></li><li> 제조 및 N -Sulfosuccinimidyl -6- (4&#39;- 아지도 -2&#39;- 니트로 페닐 아미노) 헥사 노 에이트의 사용량 분취 (설포 SANPAH는 M은 492.40 g / 몰 R). 주의 : 설포 SANPAH 눈에 심한 자극을 일으킴; 장갑과 적절한 보안경 · 안면 보호구로 처리합니다. 스토어 수령시 -20 ° C에서 이전, 분취한다. <ol><li> 설포 - SANPAH를 저장하려면 : 50 ㎎ / ㎖에서 dimethylsulfoxane (DMSO)에 설포 SANPAH을 용해. 튜브 당 20 μl를 50 마이크로 원심 분리기 튜브에 원액을 나누어지는. 드라이 아이스 나 액체 질소에 플래시 동결 -80 ° C에서 저장 (최대 가교제 효율을 유지하기위한 선택 사항이지만 선호 단계). 참고 : 분취 캘리포니아N 수개월 동안 저장 될 수있다. </li><li> 설포 - SANPAH를 사용하려면 : 나누어지는 잠시 해동 및 탈 이온수 480 μL에 희석. 즉시 사용합니다. 설포 SANPAH 물에 빠르게 가수 분해 : 따라서 신속하게 위의 단계를 모두 수행 할주의하십시오. </li></ol></li><li> 과 황산 암모늄 (M R 228.18 g / mol)을 분취의 준비. 참고 :과 황산 암모늄이 눈, 피부, 호흡기에 자극을 일으킴. 취급시 적절한 개인 보호 장비를 착용 할 것. 화학 흄 후드에서과 황산 암모늄 분말을 처리합니다. 건조하고 통풍이 잘되는 곳에 보관 분말. 분취하면, 희석 용액은 작업대에 사용될 수있다. <ol><li> / V w 10 %의 최종 황산 암모늄 농도를 달성하기 위해 증류수에 황산 암모늄을 녹인다. -20 ° C에서 나누어지는 및 저장. 사용하기 직전에 해동. </li></ol></li><li> 콜라겐 타입 I 용액의 조제. <ol><li> 그래서 주식을 희석하여 0.2 ㎎ / ㎖ 콜라겐 솔루션을 준비합니다산도 7.4의 1 배 인산 완충 식염수 (PBS)의 기술인 것. 얼음에 잠시 보관 또는 희석 즉시 사용한다. </li></ol></li></ol> 2. 하이드로 겔 준비 (그림 1 참조) <ol><li> 소수성 유리 슬라이드의 준비. <ol><li> 유리판 상에 소수성 용액의 몇 방울을 넣고 표면을 가로 질러 확산하는 휴지를 사용한다. 공기를 건조 시키과 소수성 코팅을 균일하게 다시 티슈로 닦아냅니다. 참고 : RT에서 가연성 캐비닛에 보관 소수성 솔루션입니다. 취급시 개인 보호 장비를 착용 할 것. 환기가 잘되는 곳에서 작업 할 수 있습니다. </li></ol></li><li> 유연한 플라스틱 지원 준비. <ol><li> 소수성 코팅 된 유리판의 크기에 맞게 유연한 플라스틱 지지체를 잘라. </li><li> 가볍게 같은 메스와 같은 날카로운 도구로 표면을 긁어서 유연한 플라스틱 지원의 소수성 측면을 표시합니다. 참고 : 젤 번 - 완전히 투명 - 마를스크래치 마크는가요 성 플라스틱 지지체 측을 포함하는 겔을 구별 할 수 있도록한다. 참고 : 유연한 플라스틱 지원은 소수성과 친수성 ​​측면이있다. 일단 침착, 폴리 아크릴 아미드가 영구적으로 쉽게 이후의 하이드로 겔 처리를 용이하게 플라스틱 지원의 친수성 ​​측면을 준수합니다. </li></ol></li><li> 젤 제조 예 (실시 예 5 ㎖ 볼륨 겔 전구체 용액 주어진다). 주 : 겔 초기 두께가 0.5 mm 인 경우는 5 ~ 40 ㎖의 겔을 제조하기에 충분하다. 겔 두께가 감소하는 경우는 겔을 제조 할 수있다. <ol><li> 50 ML 원뿔 튜브에 원하는 최종 농도 (표 1)의 폴리 아크릴 아미드 전구체 용액의 4972.5 μl를 놓습니다. 열린 캡 30 분 동안 탈 가스 챔버에 배치합니다. 주 : 산소 자유 라디칼 트랩으로서 작용하며, 본, 그것을 중합을 억제한다. </li><li> 과 황산 암모늄의 v / w 10 %의 25 μl를 추가 최종를 달성하기 위해 (1.3 포인트 참조)0.05 %의 황산 암모늄 농도. </li><li> N, N, N &#39;, N&#39;- 테트라 메틸 에틸렌 2.5 μl를 추가 0.5 % TEMED 최종 농도를 달성하기 위해 탈기 겔 용액 (TEMED, M은 116.24 g / 몰 R). 참고 : 저장 TEMED 실온에서 가연성 캐비닛. 적절한 개인 보호 장비를 착용 할 때 화학 흄 후드 취급 할 것. 이 높은 공기와 습기 민감로 단단히 불활성 가스 하에서 닫아 보관할 것. </li><li> 5 회 - 아래 3을 피펫 팅에 의해 부드럽게 솔루션을 섞는다. 겔 전구체 용액에 산소 확산을 피하기 위해 와동 마십시오. </li><li> 소수성 코팅 된 유리 슬라이드와 유연한 플라스틱 지원 및 샌드위치의 친수성면에 겔 용액을 피펫. 원하는 두께 (예를 들면, 0.5 mm)의 실리콘 스페이서 두 개의 슬라이드를 분리합니다. 겔화의 지표로서 사용하는 50 ㎖ 원뿔형 튜브에 폴리머 전구체 용액을 소량 (~ 100 μL)를 떠난다. 참고 :상관 스페이서가 사용될 수있다. 120 μm의 - 예를 들어, 파라 필름의 하나의 스트립 (100)의 최종 두께 팽윤 된 겔을 준다. </li><li> 중합시에도 하이드로 겔 표면을 확인하기 위해 유연한 플라스틱 지원 / 젤 샌드위치 꼭대기 다른 유리 접시를 놓습니다. </li><li> 원하는 경우에 더 적은 시간이 더 높은 중량 %의 겔에 사용될 수 있지만, 겔, 45 분 동안 중합하자. 겔 중합 된 것을 확인하기 위해, 50 ㎖ 원뿔형 튜브에 남은 용액을 관찰; 나머지 용액을 겔화 한 경우, 그때는 유리판뿐만 아니라, 겔화 개시되었음을 보인다. 그러나, 몰드의 개방 완료 조기 중합을 방지 할 수 있습니다. </li><li> 겔이 형성되면, 상부에 공유 적으로 부착 된 폴리 아크릴 아미드 겔로가요 성 플라스틱 지지체를 박리하고, 자연 건조 겔 - 측 업을 설정한다. 주 : 대류 나 가열 건조는이 공정을 촉진시킬 수있다. 가요 성 플라스틱 지지체 상에 건조 후, 겔 저장소 일 수있다무기한 거라고. <ol><li> 폴리 아크릴 아마이드 젤이 거품으로 인해 형성하지 않은 유연한 플라스틱 지원의 벌거 벗은 반점을 표시합니다. 젤은 건조되면이 유연한 플라스틱 지원 조화 ​​것으로 표시 겔 여전히 수화된다. </li></ol></li></ol></li></ol> 3. 멀티 웰 플레이트 조립, 콜라겐 코팅 및 살균 <ol><li> 멀티 웰 플레이트 어셈블리. <ol><li> 건조되면, 원하는 모양으로 PA 젤을 잘라. <ol><li> 96- 웰 플레이트를 들어, ~ 6mm 직경의 대형 구멍 펀치를 사용한다. 정사각형 또는 직사각형 모양으로 젤을 잘라 대형 종이 커터를 사용합니다. 또한, 가위를 사용합니다. </li></ol></li><li> 제조업체의 지침에 따라 96 웰 플레이트 당 PDMS의 약 500 μl를 준비합니다. 멀티 웰 플레이트의 바닥에 겔 아교, 각 웰의 중앙에 작은 물방울은 폴리 디메틸 실록산 (PDMS)의 (~ 5 μL)를 올려. 집게를 사용하면, 하나의 폴리 아크릴 아미드를 배치아래 각 웰, 유연한 플라스틱 지원 측면에서 전자 젤. PDMS는, 치료 4 시간의 최소 37 ° C에서 조립 판을 떠날 수 있도록합니다. </li></ol></li><li> 폴리 아크릴 아마이드 겔의 콜라겐 코팅. <ol><li> 전송 피펫으로 소량의 배치 - 설포 - SANPAH (7 8 μl를) 겔 표면에 균일하게 코팅 좌우로 잘 소용돌이 각 (1.2.2를 가리 키도록 참조). 설포 - SANPAH 물 안정되지 않기 때문에 신속하게 작업 할 수 있습니다. </li><li> 5 분 - (365 nm의 강도 = 37 mW의 λ = 302) 고강도 UV 램프에서 잘 플레이트를 놓습니다. 초과 설포 SANPAH을 제거하기 위해 PBS와 젤을 씻어. </li><li> 50 μL 0.2 mg을 피펫 / ㎖ 콜라겐 유형 I 솔루션을 각 웰에 (1.4 포인트 참조). 4 ℃에서 적어도 2 시간 또는 O / N RT에서 커버 플레이트를 남겨주세요. 주 : 콜라겐 코팅 프로세스 속도, 전송 피펫은 콜라겐 용액을 부가하는데 사용될 수있다. 일반적으로, 1 - 시액 2 방울을 완료하기에 충분합니다LY는 겔 표면을 커버. </li><li> 콜라겐 결합을 가능하도록 적어도 2 시간 동안 RT에서 웰 플레이트를 남겨. </li><li> 2 시간 동안 조직 배양 후드에서 과도한 콜라겐 용액을 제거하고, UV (λ = 200 ㎚) 하에서 멸균 PBS로 씻어. </li><li> 수분과 평형을 O / N (중간 컴포지션 4.1 절 참조) 완전한 매체에 젤을 만끽. 셀은 최대 2 일 동안 냉장고에 바로 저장하거나 시드에 사용합니다. </li></ol></li></ol> PA 강성 분석 4. 셀 시드 음표 : 공통 포유류 세포주 전형적인 있지만,이 섹션에 설명 된 프로토콜을 구체적 유방암 MDA-MB-231 세포주와 함께 사용된다 (도 4 및 5 참조). <ol><li> 37 ° C에서 5 분 동안 5 % 트립신 / EDTA에 노출 조직 배양 플라스크로부터 세포를 수집한다. 문화 플라스크 영역의 각 cm 2 당 트립신 / EDTA의 ~ 80 μl를 사용; 예를 들면, fo를 트립신 / EDTA 2 ㎖를 사용하여라 T25 세포 배양 플라스크. 다시 일시 원하는 최종 세포 농도로 10 % 소 태아 혈청 (FBS) 및 1 % 페니실린 / 스트렙토 마이신이 보충 된 완전 배지에 세포를 수집. 계정 배가 시간 셀뿐만 아니라 세포 분석법에 접종 될 시간의 길이를 고려하여 적절한 서브 - 융합 세포 농도를 선택한다. 추가 매체가 완전히 하이드로 겔 잠수함 충분히 있는지 확인하십시오. 참고 : 하이드로 겔이 매체에 미리 평형 되었기 때문에 충분해야 100 μL 볼륨 (3.2.6 단계를 참조하십시오). 젤 이전 단계에서 언론에 사전 평형 되었기 때문에 멀티 웰 플레이트에 사용되는 일반적인 매체 볼륨은 충분합니다. 주 : 분석은 첨부 파일에 의존하는 세포 유형에 적합한 것입니다. <ol><li> hemacytometer를 사용하여 반전 현미경 세포 수를 계산. 로드 (10) 각각의 포트에 hemacytometer 세포 현탁액 μL 및 적어도 8 사분면에서 세포 수의 평균. 를 얻으려면최종 세포 농도가 10 (4)에 의해 세포 수를 곱한다. 참고 : 각 hemacytometer 사분면에 휴대폰 번호가 20 인 경우 최저 세포 수의 결과를 얻을 수있다 - (50). </li></ol></li><li> 문화 37 ° C에서 가습 인큐베이터에서 PA 강성 분석 상 세포와 5 % CO 2. 3 일 - 매 2 미디어를 변경합니다. 5 분 동안 37 ° C에서 5 % 트립신 / EDTA에 노출 됨으로써 필요할 때 세포를 수집한다. 조직 배양 플라스크의 cm 2 당 트립신 / EDTA의 ~ 80 μl를 사용합니다. 약간 옆으로 멀티 웰 플레이트 팁과 하이드로 겔을 터치 반대로, 잘 각각의 측면 벽에 피펫 팁을 터치하여 흡인 또는 피펫 매체 : 모든 세포 조작 단계 동안, 특별한 하이드로 겔 표면을 방해하지 않도록주의하십시오. 주 : 표준 조직 배양 처리 동안 하이드로 겔의 표면을 손상하지 않도록 특별한주의하면서 제외한 세포들은 시딩에 것처럼 강성 분석법 동일한 방식으로 조작 할 수있는 상으로 접종일반 멀티 웰 플레이트. </li></ol> PA 강성 분석에 시드 세포의 5. 이미징 <ol><li> 직접 PA 강성 분석에 이미지 세포. 주 : 상관 현미경 - 반전, 형광 또는 공 촛점은 세포 이미징을 위해 사용될 수있다. 주 : 강성 분석법을 구성하는 데 사용되는가요 성 플라스틱 지지체는 완전히 투명하며 autofluoresce 또는 세포 이미징을 방해하지 않는다. 그러나,가요 성 플라스틱 지지체 자체가 투명한 경우에도, 결상 성능이 목표의 작동 거리에 의해 제한 될 것이다. 가요 성 플라스틱 지지체는 급격히 감소에 대해 높은 배율, 0.23 mm의 두께 및 10X 대물 렌즈의 작동 거리 전형적인 ~ 4mm 인을 갖는다. <ol><li> 라이브 세포 이미징의 경우, 현미경 플레이트 홀더와 이미지의 위치 PA 강성 분석. 2 시간에서 영상 세션을 유지, 이상 이미징 회 환경 챔버가 장착 된 현미경을 사용합니다. </li><li> 때 살아있는 세포 메신저노화 목표 아니다, 0.1 %의 세제를 보충 4 % 포름 알데히드 용액에 세포를 고정합니다. 2 시간 이상 동안 실온에서 세포를 고정액에 담근다. PBS로 2 회 씻어. 지정 폐기물 용기에 정착 폐기물을 폐기하십시오. 참고 : 세포는 즉시 이미지를 만들 수 있습니다 또는 최대 2 주 동안 4 ° C에서 PBS에 빠져들 저장됩니다. 주의 : 포름 알데히드는 흡입 및 접촉시 독성. 화학 흄 후드 장갑으로 처리합니다. 주 : 특정 고정 제는 일부 세포 단백질을 손상하기 때문에 세포 정착 프로토콜은 후속하는 세포 염색 및 처리 프로토콜에 기초하여 선택되어야한다. </li><li> 형광 이미징의 경우, 얼룩은 멀티 웰 플레이트에서 직접 원하는 셀 얼룩 세포를 고정. 이미지 즉시. </li></ol></li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+generating+patterned+co-cultures+with+stable+interfaces.">A simple and rapid method for generating patterned co-cultures with stable interfaces.</a> BioTechniques. 55 (1), 21-26 (2013).</li><li>Tarone, G., Galetto, G., Prat, M., Comoglio, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+molecules+and+fibronectin-mediated+cell+adhesion:+effect+of+proteolytic+digestion+of+membrane+proteins.">Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins.</a> The Journal of cell biology. 94 (1), 179-186 (1982).</li><li>Trujillo, V., Kim, J., Hayward, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creasing+instability+of+surface-attached+hydrogels.">Creasing instability of surface-attached hydrogels.</a> Soft Matter. 4 (3), 564-569 (2008).</li><li>Saha, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+creasing+instability+of+soft+polyacrylamide+cell+culture+substrates.">Surface creasing instability of soft polyacrylamide cell culture substrates.</a> Biophysical journal. 99 (12), L94-L96 (2010).</li><li>Buxboim, A., Rajagopal, K., Andre&#8217;EX, B., Discher, D. 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The authors have nothing to disclose.

원래 개발 된 전기 영동 겔은 폴리 아크릴 아미드, (28)은 현재 일상적으로 세포 형태, 운동성 및 다른 전지 특성 간의 통신 3,24,29 기판에 강성의 영향을 연구하는 세포 배양 용 기재로서 사용된다. (그림 2, 표 1도 참조 17,25,26 참조) 고분자 전구체 농도의 간단한 변화 - (300 kPa의 0.3) (1) 폴리 아크릴 아미드는 기판 강성의 조작은 신체의 모든 연부 조직의 ?...

몸에있는 대부분의 조직은 1 ~ 조달 협정 계수가 0.1 kPa 내지 부드러운 연골 100 kPa의 뇌, 아직, 체외 세포 연구에서 대부분의 조직 배양 폴리스티렌 (TCP)에 실시에 대한 범위의 탄성 계수와 부드러운 점탄성 물질이다 . 1이 강성 불일치가 크게 세포가 자신의 환경에 반응하는 방식에 영향을 미친다. 연구 성장 본체, 2,3- 줄기 세포를 포함. 그 결과 4 다양한 세포 유형의 운명에 기판의 강성의 영향을 밝히기에 따라서 전용 인 다중 하이드로 겔 강성 - 의존성 세포의 이해를 돕기 위하여 개발되어왔다 기판 강성 세포 운명에 상당한 영향을 미치고 있다는 증거가 증가하고 있지만, 폴리 아크릴 아미드 (PA), 5-7, 폴리에틸렌 글리콜 (PEG), 8,9 디메틸 실록산 (PDMS), (10) 및 알긴산 포함한 생물학. 11, 가장 연구가 실시되고 (S)의 수가 적은 소규모불가능한 명령어. 세포 유형 또는 환경 조건이 드물다의 배열을위한 기판 강성의 효과에 대한 체계적, 다차원 연구. (12) 몇몇 유망 높은 처리량 하이드로 겔 기술은 PEG 계 마이크로 어레이, 강성 마이크로로드의 직경 및 높이에 의해 변조되고, 아가 로스 겔 마이크로 비드 (14) 또는 마이크로 및 나노로드의 제조를위한 (13)의 마이크로 유체 장치를 포함하여, 개발되어있다. (15) 그러나 , 기술은 기판 정교하고 실험실의 수가 제한되어 있습니다 제조한다. 강도 변조 된 세포 반응을 포함하는 많은 연구가 구현뿐만 아니라, 영률, 즉 0.3의 생리 학적으로 중요한 범위 발휘할 만 저렴하고 간단하지 않은 폴리 아크릴 아미드 (PA) 겔 이용한다 -. 300 kPa로를 16-22 그러나 PA를 제조하는 방법을 기존 세포 배양 젤은 노동 집약적 결과적으로 준비하다작은 일괄 ared. 세포 기질로서 PA 겔의 제조와 관련된 문제의 일부가 겔을 제조 할 필요가 요구 유래한다 : 1) 산소의 부재하에 완전한 중합을 허용하기 위해, 2) 평활 한 표면과 균일 한 셀을 허용 부착 및 확산, 3) 부동 영구적 방지 세포 배양 접시의 바닥에 부착. 몇몇 그룹은 대형 일괄 적으로 세포 배양을위한 PA 젤을 생산하려고했습니다. 셈러 등. 홀 펀치 후 &quot;컷&quot;이었고, 96 웰 플레이트에 배치 PA 젤의 준비 두께의 시트. (23) 그러나이 방법은 엄격한 젤, 즉,&gt; 1 kPa의 영률, 부드러워로 제한됩니다 젤은 삭재하고 쉽게 손상 &quot;끈적 끈적한&quot;입니다. MIH가 외. 겔 직접 유리 바닥 멀티 웰 플레이트에서 중합 될 수 있도록 더 정교한 기술을 개발 하였다. <s최대&gt; (6)이 작용 유리 바닥 판에 겔 솔루션을 따르고 사용자 정의 커버 글라스 배열로 &quot;를 사이에두고&quot;에 의해 겔을 형성함으로써 달성되었다. 6 비록 매우 유망한, 약간의 가장자리 효과는 아직이 기술로 관찰되었다. 또한,이 기술은 많은 실험실뿐만 아니라 비용이 많이 드는 유리 바닥 멀티 웰 플레이트에 즉시 액세스 할 수없는 맞춤 설계 배열이 필요합니다. 이 논문은 쉽게 실험실에 의해 채택 될 수있는 멀티 웰 플레이트에서 PA 젤을 조립하는 간단하고 저렴한 방법을 설명합니다. 구충제는 PA 젤에있는 소수성 하나, 및 공유 증착에 PA 젤을 결합하는 친수성 ​​하나, - 여기, 유연한 플라스틱 지원은 두 가지 측면이있는, 사용된다. PA 겔 시트는가요 성 플라스틱 지지체에 부착 된 영구 증착이 된 후, 임의의 두께 나 강성의 겔을 처리하고 임의의 바람직한 형태로 절단 가능하게한다. 이 APPRoach는 PA 결합 액으로, 커버 글라스 또는 비용이 많이 드는 유리 바닥 멀티 웰 플레이트의 우물를 달리 상업적으로 사용할 수없는 크기의 사용자 정의 플라스틱 &#39;된 커버&#39;를 생산하고, 또한 유리 표면 - 치료의 사전 할 필요성을 미연에 방지뿐만 아니라 지루하고 시간 소모적 인 단계를 포함한다. 마지막으로, 균일 PA 겔 시트를 큰 배치로 제조하고, 몇 달 동안 탈 수화 저장 될 수있다. 요약하면, 여기에 제시된 분석은 몇 가지 측면에서 기존의 방법에 비해 개선이다. 첫째, 멀티 웰 플레이트 조립체의 프로세스는 효율적이고, 요구되는 물질의 총 비용은 낮다. 둘째, 하이드로 겔은 하나의 균질 한 겔 필름에 큰 일괄 적으로 생산된다. 마지막으로, 상업적으로 사용할 수있는 유일한 재료가 필요합니다. 분석의 유틸리티는 세포 형태에 강성 기판의 효과를 탐구하고 확산 영역으로 도시된다.

<table border="0" cellpadding="0" cellspacing="0" width="898">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Reagents</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">40% Acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0140</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">2% Bis-acrylamide</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-0142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Ammonium Persulfate</td>
			<td style="width:200px;">Bio-Rad</td>
			<td style="width:187px;">161-07000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">TEMED</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T9281</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Sulfo-SANPAH</td>
			<td style="width:200px;">Thermo Scientific</td>
			<td style="width:187px;">22589</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Collagen Type 1, from Rat tail, 3.68 mg/mL</td>
			<td style="width:200px;">BD Biosciences</td>
			<td style="width:187px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:200px;">Fisher Scientific&#160;</td>
			<td style="width:187px;">BP231-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">Hydrophobic solution - Repel Silane&#160;</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">17-1332-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:303px;">PBS (1x), pH 7.4</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:303px;">Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td>
			<td style="width:200px;">Elsworth Adhesives</td>
			<td style="width:187px;">3097358-1004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tyrpsin/EDTA (10x)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">44174</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">RPMI-1640 Medium (1x)</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30027-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Fetal Bovine Serum</td>
			<td style="width:200px;">HyClone</td>
			<td style="width:187px;">SH30073-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Penicillin Streptomycin</td>
			<td style="width:200px;">MP Biomedicals</td>
			<td style="width:187px;">1670046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Detergent - Triton-X</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Formaldehyde 37% Solution</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">F1635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:200px;">Sigma Aldrich</td>
			<td style="width:187px;">A2153</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">BSA-based cell adhesion blocking kit - ECM Cell Adhesion Array Kit</td>
			<td style="width:200px;">Chemicon International</td>
			<td style="width:187px;">ECM540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">flexible plastic support - GelBond PAG Film for Polyacrylamide Gels</td>
			<td style="width:200px;">GE Healthcare Bio-Sciences</td>
			<td style="width:187px;">309819</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Glass Plates</td>
			<td style="width:200px;">Slumpys</td>
			<td style="width:187px;">GBS4100SFSL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">50 mL Conicals</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">3181345107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">15 mL Conicals</td>
			<td style="width:200px;">FALCON</td>
			<td style="width:187px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Micro centrifuge tubes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2 mL: 02681258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">96-well plate (flat bottom)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">12565501</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:303px;">Disposable Pipettes (1 mL, 2mL, 5mL, 10mL, 25 mL, 50mL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Glass Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">5 3/4&#34;: 1367820A, 9&#34;:136786B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Tips (1-200uL, 101-1000uL)</td>
			<td style="width:200px;">Fisher Scinetific</td>
			<td style="width:187px;">2707509</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Plastic Standard Disposable Transfer Pipettes</td>
			<td style="width:200px;">Fisher Scientific</td>
			<td style="width:187px;">13-711-9D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Parafilm</td>
			<td style="width:200px;">PARAFILM&#160;</td>
			<td style="width:187px;">PM992</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Powder Free Examination Gloves</td>
			<td style="width:200px;">Quest</td>
			<td style="width:187px;">92897</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td>
			<td style="width:200px;">Grace Bio-Labs</td>
			<td style="width:187px;">JTR-S-0.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;"></td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Large/non-disposable lab equipment</td>
			<td style="width:200px;"></td>
			<td style="width:187px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:303px;">Light and Flourescent Microscope (Axiovert 200M)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">3820005619</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Microscope Software</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">AxioVision Rel. 4.8.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">UV oven</td>
			<td style="width:200px;">UVITRON</td>
			<td style="width:187px;">UV1080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum chamber/degasser</td>
			<td style="width:200px;">BelArt</td>
			<td style="width:187px;">999320237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Vacuum pump for degasser</td>
			<td style="width:200px;">KNF Lab</td>
			<td style="width:187px;">5097482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Tissue Culture Hood</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-425-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Chemical Fume Hood</td>
			<td style="width:200px;">KEWAUNEE</td>
			<td style="width:187px;">99151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Inverted Microscope (Axiovert 25)</td>
			<td style="width:200px;">Zeiss</td>
			<td style="width:187px;">663526</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Incubator</td>
			<td style="width:200px;">NUAIRE</td>
			<td style="width:187px;">NU-8500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Pipette Aid</td>
			<td style="width:200px;">Drummond Scientific Co.</td>
			<td style="width:187px;">P-76864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">Hemacytometer</td>
			<td style="width:200px;">Bright-Line</td>
			<td style="width:187px;">383684</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple polyacrylamide-based multiwell stiffness assay for the study of stiffness-dependent cell responses

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 &#8211; 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn&#8217;t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

폴리 아크릴 아미드 (PA) 하이드로 겔 널리 강성 - 의존성 세포 반응을 테스트하는 데 사용된다. 17,24을 아크릴 아미드 (A), 비스 - 아크릴 아미드 (B) 중 하나는 가장 연조직 강성 범위에 걸쳐 PA 겔을 만들 수있는 다양한 농도의 혼합함으로써 몸 - 0.3 -.. 여기에 300 kPa의 탄성 계수 (1) 그러나, 폴리 아크릴 아마이드 겔의 준비 지루한 시간이 종종 예를 들어 약물 스크리닝으로 &quot;높은 ?...

Watch this Scientific Journal Video about Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses at JoVE.com

Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

간단한 폴리 아크릴 아미드 기반 강성 의존 세포 반응의 연구를위한 멀티 웰 강성 분석

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a ...

simple-polyacrylamide-based-multiwell-stiffness-assay-for-study

Research

JoVE Journal

Bioengineering

19.4K 조회수.  Saint Louis University. Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

비디오: Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

엔지니어링된 심장 조직 이식 다음과 같은 면역 반응의 평가 (EHT)에 대한 Bioluminescence 이미징

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

연구

생체공학

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

3D Spheroids의 생성을위한 간단한 한깅 드롭 세포 배양 프로토콜

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypic 콜라겐 I 분석 : 3 차원 컨텍스트에서 셀 행동을 평가하기 위해 가단 플랫폼

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

FlexStation 3을 사용 TRP 채널 기능의 높은 처리량 검사하는 매체를위한 셀 기반의 칼슘 분석

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

에 대한 접착 주파수 분석 현장에서</em셀 - 셀 인터페이스에서 교차 Junctional 분자의 상호 작용> 속도론 해석

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Micropipette Aspiration of Substrate-attached Cells to Estimate Cell Stiffness

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell proliferation, differentiation, migration and cell-cell interactions. The two key parameters of cellular biomechanics are cellular deformability or stiffness and the ability of the cells to contract and generate force. Here we describe a quick and simple method to estimate cell stiffness by measuring the degree of membrane deformation in response to negative pressure applied by a glass micropipette to the cell surface, a technique that is called Micropipette Aspiration or Microaspiration.
Microaspiration is performed by pulling a glass capillary to create a micropipette with a very small tip (2-50 &mu;m diameter depending on the size of a cell or a tissue sample), which is then connected to a pneumatic pressure transducer and brought to a close vicinity of a cell under a microscope. When the tip of the pipette touches a cell, a step of negative pressure is applied to the pipette by the pneumatic pressure transducer generating well-defined pressure on the cell membrane. In response to pressure, the membrane is aspirated into the pipette and progressive membrane deformation or "membrane projection" into the pipette is measured as a function of time. The basic principle of this experimental approach is that the degree of membrane deformation in response to a defined mechanical force is a function of membrane stiffness. The stiffer the membrane is, the slower the rate of membrane deformation and the shorter the steady-state aspiration length.The technique can be performed on isolated cells, both in suspension and substrate-attached, large organelles, and liposomes.
Analysis is performed by comparing maximal membrane deformations achieved under a given pressure for different cell populations or experimental conditions. A "stiffness coefficient" is estimated by plotting the aspirated length of membrane deformation as a function of the applied pressure. Furthermore, the data can be further analyzed to estimate the Young's modulus of the cells (E), the most common parameter to characterize stiffness of materials. It is important to note that plasma membranes of eukaryotic cells can be viewed as a bi-component system where membrane lipid bilayer is underlied by the sub-membrane cytoskeleton and that it is the cytoskeleton that constitutes the mechanical scaffold of the membrane and dominates the deformability of the cellular envelope. This approach, therefore, allows probing the biomechanical properties of the sub-membrane cytoskeleton.

Here we describe a quick and simple method to measure cell stiffness. The general principle of this approach is to measure membrane deformation in response to well-defined negative pressure applied through a micropipette to the cell surface. This method provides a powerful tool to study biomechanical properties of substrate-attached cells.

기판에 연결된 전지의 Micropipette의 열망은 셀 강성을 추정하기 위해

Growing number of studies show that biomechanical properties of individual cells play major roles in multiple cellular functions, including cell ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

라이브 이미징 및 상해 응답의 연구에 대한 마이크로 유체 칩을 사용<em&gt; 초파리</em&gt; 애벌레

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D 상호 작용의 연구에 대한 3D 콜라겐 젤과 마이크로 채널 준비<em&gt; 생체</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

섬유에 유기 전자 장치를위한 간편하고 확장 가능한 제조 방법

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

화학 유인 그라데이션에 대 한 다중 세포 반응의 3D 분석

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...