This study was supported by a Merit Award from the U.S. Department of Veterans Affairs, and an NIH grant R01CA1385282522717 to RW.

재료 및 시약 1. 준비 <ol><li> 레이블 조직 배양의 각 웰은 각각의 세포주 또는 조건에 맞게 6 웰 플레이트 치료가 연구되고. </li><li> 분말 매체의 1g 및 50 ㎖의 최종 부피로 탈 이온수에 탄산 수​​소 나트륨 0.2 g을 용해시켜 배 세포 배양 배지를 준비한다. </li><li> 소독하는 0.2 μm의 필터를 통해이 매체를 전달합니다. </li><li> 관심의 세포 라인의 일반 문화에 필요한 추가 구성 요소를 추가합니다. 예를 들어, 10 % FBS 및 1 % 페니실린 / 스트렙토 마이신 용액으로 보충 된 RPMI 1640 배지에서 CMT 167 세포주 성장. 사용하는 뜨거운 물을 욕조에 37 ° C 따뜻한 매체는 이전에. </li><li> 만약 관심 세포주의 정상 세포 배양 용 마찬가지로 별도로 배속 세포 배양 배지를 준비한다. </li><li> 탈 이온수의 고귀한 한천 100 ml의 1 g을 추가하여 1 % 귀족 한천을 준비합니다. 참고 : 빛나는 한천 혼자 교반 완전히 용해되지 않습니다. </li><li> 준비0.6 % 탈 이온수 100 ㎖에 귀금속 한천 0.6 g을 첨가하여 한천 노블. 두 한천 용액은 장기간 저장을 위해 폐쇄 가능한 뚜껑 100 ml의 유리 병에서 만들어 질 수있다. </li><li> 소독 고귀한 한천 혼합물을 압력솥. 이러한 혼합물은 사전에 4 ° C에서 저장하지만 한천이 완전히 용해 될 때까지 실험시 재가열되어야 할 수있다. </li><li> 1000 ml의 최종 부피로 H 2 O에 PO 4 HPO 4 1X PBS (8g의 NaCl, 0.2 g의 KCl, 1.44 g이 나트륨에 1 ㎎ / ㎖ 원액함으로써 nitroblue 테트라 졸륨 클로라이드 용액을 준비하고, 0.24 g KH 2 ). 이 콜로니 얼룩이 실험의 마지막에 사용된다. </li></ol> 한천의 하단 레이어 2. 도금 <ol><li> 약 1-2 분 동안 1 % 귀족 한천과 전자 레인지의 병 뚜껑을 풉니 다. 전자 레인지에 가열하면서, 끓어 방지하기 위해 밀접하게 솔루션을 모니터링 할 수 있습니다. 간헐적으로 혼합하면서 가열을 계속,한천이 완전히 용해 될 때까지 용액을 분명하다. 참고 : 가열 후 플라스크를 처리하기 위해 내열 장갑을 사용합니다. 그렇지 않을 경우 화상 또는 심각한 부상을 초래할 수 있습니다. </li><li> 뜨거운 수돗물 (42 ° C)로 가득 얼음 양동이에 녹아 한천 솔루션과 미리 예열 배 배양 배지를 놓습니다. 또한 뜨거운 물을 얼음 통에 튜브 홀더에 50 ML 원뿔 튜브를 배치합니다. 다음 단계는 세포 배양 후드에 버킷을 전송합니다. </li><li> 한천의 맨 아래 층의 경우, 6 웰 플레이트 잘 당 한천의 믹스와 매체의 1.5 ml의가 필요합니다. </li><li> 혼합물의 적절한 양을 보장하기 위해, 각각의 6- 웰 플레이트 12 ㎖가 총을 준비한다. </li><li> 50 ㎖ 원뿔형 튜브에 배양 배지 6 ㎖의 1 % 귀금속 한천 용액, 6㎖를 첨가하여 시작. </li><li> 혼합 원뿔 튜브를 여러 번 반전. 활발한 속도로 작업 부드러운 한천의 조기 경화를 방지 할 수 있습니다. </li><li> 5 ml의 혈청 학적 파이로 혼합물의 약 5.5 ml의를 그립니다pette. </li><li> 기포가 아니라 각에이​​ 혼합물의 1.5 ml의 증착하기 전에 피펫 컬럼의 상단까지 상승 할 수 있습니다. 판 우물에 공기 방울의 증착을 방지하기 위해주의해야합니다. </li><li> 플레이트를 덮고 한천 혼합물을 30 분 동안 세포 배양 후드에서 실온에서 고화 할 수있다. </li></ol> 3. 도금 한천 함유 세포의 상층 <ol><li> 한천의 하층이 응고되면, 상층의 준비를 시작한다. </li><li> (트립신 1 ml의 예 : 매체의 4를 가하여) 배지에서 5 15 ML 원뿔 튜브로 첫째, 트립신에 의해 수확 세포와 그들에게 일을 희석. </li><li> 세포를 세어 이때 최종 세포 현탁액을 제조 웰 당 필요한 세포의 수를 계산한다. 이 숫자는 세포의 종류에 따라 달라질 수 있습니다. 5000 세포 / 웰 같은 시작점을 사용하고 필요에 따라 조정한다. 이 세포 수의 경우, 즉, 각 아 (6667 세포 / ml의 세포 현탁액을 제조 할난이 서스펜션의 0.75 mL 및 1.5 ml의 전체 볼륨에 대한 한천 0.75 ㎖를 받게됩니다; 5000의 최종 총 세포 수)가 2 : 세포 농도는 1 희석한다. </li><li> 6- 웰 플레이트의 웰 당 필요한 세포 현탁액의 부피는 다시 1.5 ml의 것이다. 6 웰 플레이트 당 12 ml의 총 추가 세포 현탁액을 준비합니다. </li><li> 상기와 전자 레인지의 0.6 % 한천 솔루션을 녹여 튜브 홀더 50 ML 원뿔 튜브와 단계 3.3에서 최종 세포 현탁액과 함께 뜨거운 물을 포함하는 얼음 양동이에 배치합니다. </li><li> 후속 단계에 대한 세포 배양 후드에 녹은 0.6 % 한천과 얼음 통을 전송합니다. </li><li> 6- 웰 플레이트 당 12 ml의 총 부피를 조제 : 1 비율 (1)에 0.6 % 한천 및 세포 현탁액을 섞는다. 1.5 ml를 웰​​ 당 요구되지만 여분 위와 이루어져야한다. </li><li> 피펫 50 ML 원뿔 튜브에 세포 현탁액의 6 ml의. </li><li> 이어서, 튜브에 0.6 % 한천 (6)를 가하여. 참고 :이 혼합물의 온도를해야합니다조기 경화를 방지하고 세포 생존을 최대화하기 위해 약 42 ° C를 유지했다. </li><li> 피펫 세포를 배포 할 수있는이 혼합물을 2 ~ 3 배 빠르게 작동 후 5 ㎖의 혈청 학적 피펫으로 혼합물의 5.5 ml의를 그립니다. </li><li> 기포가 아니라 각에이​​ 혼합물의 1.5 ml의 증착하기 전에 피펫 컬럼의 상단까지 상승 할 수 있습니다. 판 우물에 공기 방울의 증착을 방지하기 위해주의해야합니다. </li><li> 셀 / 한천 혼합물을 37 ° C 가습 세포 배양 용 인큐베이터에 배치하기 전에 30 분 동안 세포 배양 후드에서 실온에서 고화하도록 허용. </li><li> 적절한 콜로니 형성에 필요한 시간은 약 이십일일 전형적 각 세포주에 대해 변화한다. </li><li> 성장 배지의 층은 탈수를 방지하기 위해 한천의 상층에 걸쳐 유지되어야한다. 회씩 첨가 배지 100 ㎕이 목적을 위해 충분하다. </li></ol> 4. 플레이트를 염색하고 계산 식민지 <ol><li> 200을 첨가하여 스테인 셀 _6 웰, 37 ° C에서 밤새 배양 접시 당 nitroblue 테트라 졸륨 클로라이드 용액 리터. </li><li> 콜로니 염색되면, 이미 저를 사용하여 웰의 사진 촬영과 화상 해석 소프트웨어를 사용하여 콜로니를 카운트. </li></ol>

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The authors have nothing to disclose.

시그널링 단백질의 종양 억제 기능의 확인 시험 관내에서 곤란하다. 이 속성을 조사하기 위해 사용할 수있는 가장 엄격한 분석 중 하나는 부드러운 한천 콜로니 형성 분석이다. 여기서는 안정적으로 그 부모 CMT167 세포주에 비해 Wnt7a 및 Fzd9 과발현 뮤린 폐 암종 세포주를 사용하여 연질 한천 콜로니 형성 분석을 보여왔다. 부드러운 한천 분석에 대해 고려해야 할 몇 가?...

소프트 한천 콜로니 형성 분석은 널리 시험 관내 세포 변형을 평가하는데 사용되는 기술이다. 역사적으로, 퍽 등의 문헌에 의해 기술. 1956 년 다른 분석법, 클론 원성 분석은, 콜로니 하나를 형성하는 세포의 능력을 평가하는 데 사용 하였다. 이 기술에서, 세포는 배양 접시 상에 분산시키고, 필요한 성장 인자를 제공하기위한 &#39;피더&#39;세포 조정 배지의 존재하에 성장. 이 기술의 한계는 단지 콜로니 형성에 관한 정보를 제공하는 것이 었습니다. 정상 세포 인해 anoikis이 호출 아폽토시스 죽음의 특정 유형으로, 앵커리지 - 독립된 성장이 방지된다. 그러나, 형질 전환 된 세포는 기판에 결합하지 않고 성장 능력과 격차가있다. 이 개념을 활용하기 위해, 연구진은 부드러운 한천 콜로니 형성 분석을 개발했다. 소프트 한천 콜로니 형성 분석은 이후 SP를 해결하기 위해,보다 최근에는, 개질 된ecific 필요가있다. 하나의 변화는 높은 처리량 콜로니 카운팅 있도록 형광 염료의 혼입을 포함한다. 또 다른 변화는 단백질 또는 DNA 샘플이 필요할 때 콜로니 형성 후 생존 세포의 검색을 허용하기 위해 전문화 된 한천 용액의 사용을 포함한다. 전통적인 소프트 한천 콜로니 형성 어 세이에서, 세포는 또한 세포 배양 배지와 혼합 된 연질 한천, 다른 층에 달려 세포 배양 배지와 혼합 된 연질 한천 층에서 성장하지만, 한천의 높은 농도를 함유한다. 이 배양 접시에 부착 세포를 방지하면서도 가시 콜로니를 형성하는 형질 전환 세포를 허용한다. 이 기술 뒤에 근거는 정상 세포의 성장과 분할 할 수 있도록 세포 외 기질 접촉 세포에 의존한다는 것입니다. 반대로, 형질 전환 된 세포의 성장과 무관하게 항상 주변 환경 분할하는 능력을 가지고있다. 따라서, 앵커리지 독립적 인 방식으로 식민지를 형성 할 수 세포는 C했다onsidered은 변형과 암을 유발한다. 이 방법의 전반적인 목표는 반 정량적 엄격한 방식으로 세포에서이 기능을 측정하는 것입니다. Wnt 신호 전달 경로는 배아 발생에서 중요한 종종 종양 3-6에서 해제 조절된다. Wnt 신호와 관련된 여러 경로가 있습니다. 정식 경로는 전사 보조 활성화 베타 카테닌 (β-catenin)에 미치는 영향을 통해 Wnt 신호 및 다운 스트림 유전자 전사 조절을 포함한다. Wnts는 예를 들어, 여러 비정규 경로를 통해 신호, 소포체 (8)에서 칼슘의 방출을 조절하는 세포 골격 구조 (7)에 관련된 요소를 조절하는 평면 셀 극성 통로, 그리고이 Wnt 칼슘 통로. 이 Wnt 리간드 Frizzled 수용체 결합을 통해 활성을 발휘한다. 여러 Wnts 폐암에서 상향 조절되는 것으로 도시 하였지만, Wnt7a 비 스말에서 하향 조절되는 것으로 밝혀졌다프로모터 메틸화 내지 9 리터 세포 폐암. Wnt7a가 아닌 정식 경로를 통해 종양 억제로 Fzd9과 행위를 결합한다. 이 Wnt-7a 및 FZD-9의 복원은 비 - 소세포 폐암 세포 (10)의 성장을 억제한다. Wnt7a / Fzd9의 효과 ERK-5, 차례로, 페 록시 좀 증식 제 - 활성화 수용체 γ를 활성화 (PPARγ) 11,12의 활성화를 통해 매개된다. 여기서는 Wnt7a 및 Fzd9 과발현은 뮤린 폐 암종 세포주의 앵커리지 - 독립된 성장의 억제 결과 것을 보여준다. 쥐의 CMT167 세포는 C57BL / lcrf 마우스 (13)의 폐암에서 파생 된 안정적 Wnt7A과 Fzd9로 형질 감염시켰다. Wnt7A 및 Fzd9의 과발현이 양적-PCR (Q-PCR) 및 Wnt7A과 Fzd9 과발현의 기능에 의해 확인 하였다는 PPARγ의 다운 스트림 활성화를 통해 확인되었다.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Cancer Cell Line of Interest</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">10032302</td>
			<td>CMT-167 Cells</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Powdered RPMI 1640 Medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">31800-089</td>
			<td style="width:167px;">Used to prepare 2X cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Liquid RPMI 1650 Medium</td>
			<td style="width:71px;">Cellgro</td>
			<td style="width:119px;">10-040-CV</td>
			<td style="width:167px;">Referred to as 1X cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">HyClone</td>
			<td style="width:119px;">SH30910.03</td>
			<td style="width:167px;">Used to supplement cell culture medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:71px;">CellGro</td>
			<td style="width:119px;">30-002-Cl</td>
			<td style="width:167px;">Used in cell culture medium.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Difco Noble Agar</td>
			<td style="width:71px;">BD Biosciences</td>
			<td style="width:119px;">214230</td>
			<td style="width:167px;">Used to prepare 1.0% and 0.6% Agar.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sodium Bicarbonate</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP-328-1</td>
			<td style="width:167px;">Used in 2X cell culture medium.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Trypsin</td>
			<td style="width:71px;">Cellgro</td>
			<td style="width:119px;">25-050-Cl</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Sterile Bottle-Top Filters</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">09-761-126</td>
			<td style="width:167px;">Used to sterile filter 2X medium.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Lipofectamine Reagent</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">18324-020</td>
			<td style="width:167px;">Used in PPAR-RE luciferase assay.</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">6-well Plates Tissue-culture Treated</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">37 degree C/5% CO2 Incubator</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Chemi-Doc Imager</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to take pictures of colonies.&#160;&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Quantity One Software</td>
			<td style="width:71px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Used to count cell colonies.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15mL Conical Tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">50mL Conical Tubes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">250mL Erlenmeyer Flasks</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Microwave</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">5mL Serological Pipettes</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pipette Aid</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Micropipette</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Hemacytometer w/ cover slip</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Pipette Tips</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Inverted Light Microscope</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Centrifuge</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Heat-Resistant Gloves</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Saran Wrap</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice Bucket</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the soft agar colony formation assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

우리 소프트 한천 콜로니 형성 분석으로 도시 된 바와 같이 CMT167 세포 Wnt7A과 Fzd9의 발현은 종양 억제에 효과적이다. 예비, 우리는 Wnt7A과 Fzd9의 mRNA가 CMT167 세포에서 낮은 수준에서 표현되는 것을 보여주기 위해 Q-PCR을 사용했다. MLE-12 세포, SV40 형질 전환 쥐의 폐 상피 세포주 (도 1)과 비교할 때 CMT167 세포는 내인성 Wnt7A과 Fzd9 낮은 수준을 보였다. 우리는 두 개의 레트로 바이러스 과발?...

Watch this Scientific Journal Video about The Soft Agar Colony Formation Assay at JoVE.com

The Soft Agar Colony Formation Assay

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

소프트 한천 콜로니 형성 분석

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Research

JoVE Journal

Biology

110.6K 조회수.  University of Illinois at Chicago. The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

비디오: The Soft Agar Colony Formation Assay

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

S. cerevisiae의의 접착 및 아가르 침략을위한 분석

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

연구

생물학

Supported Planar Bilayers for the Formation of Study of Immunological Synapses and Kinapse

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions between lymphocytes and antigen presenting cells, we use Ni2+-chelating lipids to anchor recombinant cell adhesion and MHC proteins to the upper leaflet of a bilayer with poly-histidine tags. Planar bilayers are generated by preparing lipid, treating the glass surfaces where the bilayer will form, and then forming the bilayer in a specialized chamber containing a flow-cell where the lymphocytes will be added.  Then, bilayers are charged with Ni and his-tagged recombinant proteins are added.  Finally,  lymphocytes are injected into the flow cell and TIRF microscopy can be used to image synapse formation and the mechanisms that control T cell locomotion, sites of receptor sorting,  and sites of receptor degradation.

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse. Here, we show methods for anchoring cell adhesion proteins known to modulate synapse formation to the upper leaflet of the lipid bilyer and visualize synapse formation using TIRF microscopy.

면역 시냅스와 Kinapse 연구의 형성을위한 지원 평면 Bilayers

Supported planar bilayers are powerful tools that can be used to model the molecular interactions in an immunological synapse.   To mimic the interactions ...

Isolating Stem Cells from Soft Musculoskeletal Tissues

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of excitement for treating injured and diseased tissues due to their impressive capabilities to undergo multi-lineage cell differentiation and their self-renewal ability. Most importantly, these qualities have made them advantageous for use in autologous cell transplantation therapies. The current protocol will introduce the readers to the modified preplate technique where soft tissues of the musculoskeletal system, e.g. tendon and muscle, are 1st enzymatically dissociated and then placed in collagen coated flasks with medium. The supernatant, which is composed of medium and the remaining floating cells, is serially transferred daily to new flasks. The stem cells are the slowest to adhere to the flasks which is usually takes 5-7 days (serial transfers or preplates) . By using this technique, adult stem cells present in these tissues can be easily harvested through fairly non-invasive procedures.

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.

소프트 Musculoskeletal 조직에서 줄기 세포를 분리

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

RhoC GTPase Activation Assay

RhoC GTPase has 91% homology to RhoA GTPase. Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed. However, RhoC GTPase is expressed in metastatic cancer cells at relatively low levels. Therefore, few RhoC-specific reagents have been developed. We have adapted a Rho activation assay to detect RhoC GTPase. This technique utilizes a GST-Rho binding domain fusion protein to pull out active RhoC GTPase. In addition, we can harvest total protein at the beginning of the assay to determine levels of total (GTP and GDP bound) RhoC GTPase. This allows for the determination of active versus total RhoC GTPase in the cell. Several commercial versions of this procedure have been developed however, the commercial kits are optimized for RhoA GTPase and typically do not work well for RhoC GTPase. Parts of the assay have been modified as well as development of a RhoC-specific antibody.

This protocol utilizes a pull down assay to determine the levels of active RhoC GTPase within cells.

RhoC GTPase 활성화 분석

RhoC GTPase has 91% homology to RhoA GTPase.  Because of its prevalence in cells, many reagents and techniques for RhoA GTPase have been developed.  ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

효모 콜로니 임베딩 방법

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic 분석 : 자기편 셀

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

종양 세포의 Vasculogenic 활동을 평가 Matrigel 기반 튜브 형성 분석

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a biofilm, bacterial cells often exhibit altered physiology, including enhanced resistance to antibiotics and other environmental stresses 1. Additionally, biofilms can play important roles in host-microbe interactions. Biofilms develop when bacteria transition from individual, planktonic cells to form complex, multi-cellular communities 2. In the laboratory, biofilms are studied by assessing the development of specific biofilm phenotypes. A common biofilm phenotype involves the formation of wrinkled or rugose bacterial colonies on solid agar media 3. Wrinkled colony formation provides a particularly simple and useful means to identify and characterize bacterial strains exhibiting altered biofilm phenotypes, and to investigate environmental conditions that impact biofilm formation. Wrinkled colony formation serves as an indicator of biofilm formation in a variety of bacteria, including both Gram-positive bacteria, such as Bacillus subtilis 4, and Gram-negative bacteria, such as Vibrio cholerae 5, Vibrio parahaemolyticus 6, Pseudomonas aeruginosa 7, and Vibrio fischeri 8.
The marine bacterium V. fischeri has become a model for biofilm formation due to the critical role of biofilms during host colonization: biofilms produced by V. fischeri promote its colonization of the Hawaiian bobtail squid Euprymna scolopes 8-10. Importantly, biofilm phenotypes observed in vitro correlate with the ability of V. fischeri cells to effectively colonize host animals: strains impaired for biofilm formation in vitro possess a colonization defect 9,11, while strains exhibiting increased biofilm phenotypes are enhanced for colonization 8,12. V. fischeri therefore provides a simple model system to assess the mechanisms by which bacteria regulate biofilm formation and how biofilms impact host colonization.
In this report, we describe a semi-quantitative method to assess biofilm formation using V. fischeri as a model system. This method involves the careful spotting of bacterial cultures at defined concentrations and volumes onto solid agar media; a spotted culture is synonymous to a single bacterial colony. This 'spotted culture' technique can be utilized to compare gross biofilm phenotypes at single, specified time-points (end-point assays), or to identify and characterize subtle biofilm phenotypes through time-course assays of biofilm development and measurements of the colony diameter, which is influenced by biofilm formation. Thus, this technique provides a semi-quantitative analysis of biofilm formation, permitting evaluation of the timing and patterning of wrinkled colony development and the relative size of the developing structure, characteristics that extend beyond the simple overall morphology.

We provide a simple, semi-quantitative method to investigate biofilm formation in vitro. This method takes advantage of the Zeiss stemi 2000-C Dissecting Microscope (with camera attachment) to monitor both the timing and pattern of biofilm formation, as assessed by the development of wrinkled colonies.

주름 식민지 개발을 사용하여 Biofilm 형성을 평가하기 위해 세미 양적 접근

Biofilms, or surface-attached communities of cells encapsulated in an extracellular matrix, represent a common lifestyle for many bacteria. Within a ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

에 대한 내피 세포의 관 형성의 분석<em&gt; 체외</em&gt; 혈관 신생 연구

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...