이 연구는 Fondazione Bambino Gesù와 Ricerca Corrente (이탈리아 보건부)의 C.C. 보조금으로 지원되었습니다.  엔리코 베르티니 박사(신경과학과, 신경근 및 신경퇴행성 질환 부서, 밤비노 제수 어린이 연구 병원 분자 의학 연구실), 스테파니아 페트리니 박사(공초점 현미경 핵심 시설, 연구 실험실, 밤비노 제수 어린이 연구 병원), 줄리아 페리콜리(종양혈액학과, 유전자 및 세포 치료과, 밤비노 제수 어린이 연구 병원) Roberta Ferretti(종양 혈액학, 유전자 및 세포 치료학과, 어린이 연구 병원 밤비노 제수)는 과학적 토론과 기술적 도움을 제공합니다. 마리아 빈치는 "Children with Cancer UK 펠로우십"을 수상했습니다.

1. 96웰 플레이트 코팅
참고: 동일한 플레이트에서 다른 코팅을 테스트했지만 별도의 웰에서 테스트했습니다( 보충 파일 참조).
<ol><li>매트리겔을 DMEM에서 1:100으로 희석한다. 96웰 플레이트에 웰당 100μL를 추가하고 실온에서 1시간 동안 배양합니다. 그런 다음 용액을 제거하고 100μL의 DMEM으로 웰을 두 번 세척합니다.</li>
	<li>PBS (칼슘 및 마그네슘 포함)에 희석 된 라미닌 (20 μg / mL, LN-521). 웰에 100 μL를 첨가하고 4°C에서 밤새 배양한다. 다음날 세포를 파종하기 전에 DMEM으로 두 번 세척하십시오.</li>
	<li>희석 완충액에 비트로넥틴(10μg/mL)을 희석합니다. 96웰 플레이트에 웰당 100μL를 추가하고 실온에서 1시간 동안 배양한다. 세포를 도금하기 전에 PBS (칼슘과 마그네슘이없는)로 우물을 씻으십시오.</li>
	<li>HU-피브로넥틴(30μg/mL)을ddH2O에 희석하고 웰에 100μL를 넣고 실온에서 45분 동안 배양합니다. 그런 다음 세포를 파종하기 전에 배지로 웰을 씻으십시오.</li>
</ol>2. 배양에서 iPSC의 유지 관리
참고: iPSC는 상업적으로 구입했습니다. iPSC는 건강한 인간 섬유아세포에서 파생되었으며 에피솜 기술을 사용하여 재프로그래밍되었습니다.
<ol><li>-80°C 냉동고 또는 액체 질소로부터, 동결보존된 iPSCs를 37°C 수조에서 해동시킨다. 생물학적 안전 캐비닛으로 옮기기 전에 세포가 들어있는 바이알을 70 % 에탄올로 청소하십시오.</li>
	<li>15mL 멸균 원뿔형 튜브에서 1000μL 피펫으로 5mL의 사전 예열된 세포 배양 배지(예: mTeSR1)에 세포 현탁액을 한 방울씩 추가합니다.</li>
	<li>실온(RT)에서 5분 동안 304 x g 의 셀을 원심분리합니다.</li>
	<li>배지를 제거하고 세포 펠릿을 4mL의 세포 배양 배지에 재현탁합니다.</li>
	<li>세포 현탁액을 6웰 플레이트(6웰 세포 배양 접시당5 iPSC 10개)의 2개 웰에 플레이팅하고, 여기서 마우스 배아 섬유아세포(MEF)가 이전에 플레이팅되었습니다. DMEM (10 % 태아 소 혈청, 1 % L- 글루타민 및 1 % 페니실린-스트렙토 마이신 함유)에서 2.4 x 104 / cm2 의 밀도로 iPSC를 도금하기 이틀 전에 MEF를 시드합니다.</li>
	<li>파종 후 세포 배지에 10μM의 ROCK 억제제 Y-27632를 보충합니다.</li>
	<li>처음 4-5주 동안 MEF에서 iPSC를 성장시킨 다음 mTeSR1에서 관심 있는 코팅 중 하나(1단계 및 표 1 참조)를 사용하여 피더 프리 조건(MEF 프리 조건)에서 성장시킵니다.</li>
	<li>iPSC가 70-80% 합류점일 때 RT에서 3-5분 동안 0.5mM EDTA 처리를 사용하여 1:4 통과합니다. 6웰 플레이트의 경우 0.5mM EDTA 1mL를 추가합니다(또는 다른 유형의 플레이트의 경우 비례 양). 피더가없는 조건에서 새 웰로 옮기고 37 ° C, 5 % CO2, 20 % O2에서 배양합니다.</li>
	<li>매일 새로운 mTeSR1로 배지를 교체하고 2일마다 세포를 분할합니다.</li>
</ol>3. 세포 합류의 특성 분석
<ol><li>실험을 위해 96웰 플레이트를 사용하십시오.</li>
	<li>MEF가 계대배양되지 않았는지 확인하기 위해 피더가 없는 상태에서 최소 1개월 배양한 후 웰당 10,000개의 세포를 시드합니다. 일회용 계수 슬라이드를 사용하여 광학 현미경으로 세포를 계수하십시오.</li>
	<li>실험을 세 번 수행하십시오. 따라서 3 개의 웰에서 각 파종 조건을 테스트하십시오.</li>
	<li>명시야 모드에서 세포분석기를 사용하여 파종 후 1일째부터 자동 이미지 획득을 수행합니다. 5일 동안 24시간마다 자동 이미지 획득을 수행합니다. 실험 파라미터에 대한 자세한 내용은 보충 파일을 참조하십시오.</li>
	<li>자동 대비 및 자동 노출을 사용하여 셀을 더 잘 시각화합니다.</li>
	<li>합류 분석에 대한 분석 설정( 보충 파일 참조)을 웰당 60%, 80% 및 100%의 마스크를 적용하여 웰 경계의 빛 굴절로 인한 초점 변화를 평가합니다. 위에서 언급한 다른 마스크 분석 설정을 사용하여 각 시점에서 세포 합류를 분석합니다.</li>
</ol>4. 통계 분석
<ol><li>정량적 결과를 평균(SEM)의 표준 오차± 평균으로 보고합니다.</li>
	<li>서로 다른 코팅 조건의 전반적인 차이를 비교하려면 동일한 표본을 사용하여 데이터를 얻고 스튜던트 쌍 표본 t-검정을 수행합니다. 0.05 미만의 P 값은 통계적으로 유의한 것으로 간주되며 보고된 모든 p-값은 양측입니다.</li>
</ol>5. 세포골격 마이크로필라멘트의 특성 분석
<ol><li>RT에서 10분 동안 PBS 중 4% 파라포름알데히드(4% PFA)로 세포를 고정한 후 PBS에서 2회 세척(총 10분)합니다.</li>
	<li>100 μL의 블로킹 용액 (PBS 중 5 % BSA, 0.1 % 트리톤으로 구성)을 RT에서 1 시간 동안 각 웰에 첨가하십시오.</li>
	<li>블로킹 용액을 제거하고 샘플을 PBS로 10분 동안 두 번 세척한다.</li>
	<li>샘플당 100μL의 팔로이딘-접합체 작업 용액을 추가하고 RT에서 1시간 동안 배양합니다.</li>
	<li>PBS로 세포를 두 번 세척합니다 (RT에서 10 분).</li>
	<li>Hoechst 33342로 핵을 염색하고 RT에서 10분 동안 PBS에서 1:10000으로 희석합니다.</li>
	<li>Hoechst 용액을 제거하고 매번 10분 동안 PBS로 세포를 두 번 세척합니다.</li>
	<li>샘플을 H2O로 세척하고 화학 후드 아래에서 건조시킵니다.</li>
	<li>100μL의 장착 매체(예: PBS:글리세롤, 1:1)를 추가하여 세포를 덮고 샘플의 형광을 보존합니다.</li>
	<li>백색광 레이저(WLL) 소스와 405nm 다이오드 레이저가 장착된 레이저 스캐닝 컨포칼 현미경으로 Ex/Em 493/517nm에서 세포를 관찰합니다. HC PLAPO 40x 오일 이멀젼 대물렌즈를 사용하여 순차적 컨포칼 이미지를 획득합니다. 검사된 모든 샘플에 대해 동일한 레이저 출력, 빔 스플리터, 필터 설정, 핀홀 직경 및 스캔 모드를 사용합니다.</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tightening+the+connections+between+cadherins+and+fibronectin+matrix.">Tightening the connections between cadherins and fibronectin matrix.</a> Developmental Cell. 16 (3), 327-328 (2009).</li><li>Villa-Diaz, L. G., Ross, A. M., Lahann, J., Krebsbach, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+The+evolution+of+human+pluripotent+stem+cell+culture:+from+feeder+cells+to+synthetic+coatings.">Concise review: The evolution of human pluripotent stem cell culture: from feeder cells to synthetic coatings.</a> Stem Cells. 31 (1), 1-7 (2013).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126 (4), 677-689 (2006).</li><li>Vigilante, A., 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=Identifying+Extrinsic+versus+Intrinsic+Drivers+of+Variation+in+Cell+Behavior+in+Human+iPSC+Lines+from+Healthy+Donors.">Identifying Extrinsic versus Intrinsic Drivers of Variation in Cell Behavior in Human iPSC Lines from Healthy Donors.</a> Cell Reports. 26 (8), 2078-2087 (2019).</li></ol>

저자는 공개 할 것이 없습니다.

질병 모델링 및 향후 약물 스크리닝에 iPSC를 사용하고 정밀 의학에 적용 할 수 있기 때문에 iPSC는 관련성이 높은 기술이되며 이러한 이유로 배아 줄기 세포의 생리적 상황과 더 유사한 체외 배양 조건을 명확하게 이해할 필요가 있다고 생각합니다. 이러한 맥락에서 우리는 세포가 건강하고 미분화 상태를 유지할 수 있는 조건을 이해하기 위해 야생형 iPSC를 사용하여 다양한 ECM 코팅을 테스트했습?...

유도 만능 줄기 세포 (iPSC)는 체세포에서 얻어지며 다른 세포 유형으로 분화 될 수 있습니다. 그들은 종종 질병 발병 기전을 모델링하거나 약물 스크리닝을 수행하는 시스템으로 사용되며 개인화 된 의학의 맥락에서 사용될 수있는 잠재력을 제공합니다. iPSC는 큰 잠재력을 가지고 있기 때문에 신뢰할 수 있는 모델 시스템으로 사용하기 위해 완전히 특성화하는 것이 중요합니다. 우리는 이전에 저산소 환경에서 iPSC를 성장시키는 것의 중요성을 보여주었습니다.이 세포는 해당 과정에 의존하고 호기성 환경은 산화 환원 불균형을 유발할 수 있습니다1. iPSC는 또한 다른 배양 조건, 특히 세포 외 환경에 취약합니다. 배양 조건의 최적화는 그들을 건강하고 증식시키는 핵심 문제입니다. 건강한 iPSC 배양은 일반적으로 특정 인간 장애 또는 세포 과정의 분자, 세포 및 기능적 특징을 이해하는 데 사용되는 모델의 종말점인 건강한 분화 세포로 이어질 것입니다.
이 연구에서는 간단한 프로토콜을 사용하여 별도의 웰에서 서로 다른 코팅 조건을 사용하여 iPSC의 합류를 테스트했습니다. iPSC가 제대로 부착되기 위해서는 쥐 배아 섬유아세포(MEF)의 영양층이 필요하지만 iPSC와 MEF가 공존하면 두 개의 세포 집단이 존재하기 때문에 RNA 또는 단백질 추출과 같은 분석을 수행하기가 어렵습니다. 피더 층을 피하기 위해 세포 외 기질 (ECM)에 속하는 다양한 단백질을 사용하여 천연 세포 틈새를 재현하고 피더가없는 iPSC 배양을 가졌습니다. 특히, Matrigel은 엥겔브레스-홀름-스웜(EHS) 마우스 육종에서 추출한 가용화된 기저막 제제로, 세포외 기질 단백질(즉, 라미닌, 콜라겐 IV, 헤파란 설페이트 프로테오글리칸, 엔탁틴/니도겐 및 성장 인자)이 풍부합니다2,3. 다른 사용 된 코팅 조건은 대신 ECM을 구축하는 데 관련성이 알려진 정제 된 단백질입니다 : 라미닌 -521은 배아의 내부 세포 덩어리에서 인간 다 능성 줄기 세포 (hPSC)에 의해 분비되는 것으로 알려져 있으며 출생 후 신체에서 가장 흔한 라미닌 중 하나입니다 4,5,6,7,8,9, 10,11; 비트로넥틴은 hPSC 12,13,14,15,16의 성장 및 분화를 지원하는 것으로 알려진 이종 무함유 세포 배양 매트릭스입니다. 피브로넥틴은 척추동물의 발달과 만능 상태의 배아 줄기세포의 부착 및 유지에 중요한 ECM 단백질이다 17,18,19,20,21,22,23,24,25. 다양한 코팅 조건을 사용할 수 있기 때문에 iPSC의 합류에 미치는 영향 측면에서 비교합니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">10 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td style="width:281px;">Corning</td>
			<td style="width:77px;">4488</td>
			<td style="width:176px;">Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 mL high-clarity polypropylene (PP) conical centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td>Tool</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (With Ca2+; Mg2+)</td>
			<td>Thermofisher</td>
			<td>14040133</td>
			<td>Medium</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">1x PBS (without Ca2+; Mg2+)</td>
			<td>Euroclone</td>
			<td>ECB4004L</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL Stripette Serological Pipets, Polystyrene, Individually Paper/Plastic Wrapped, Sterile</td>
			<td>Corning</td>
			<td>4487</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture microplate, 96 WELL, PS, F-Bottom</td>
			<td>Greiner Bio One</td>
			<td>655090</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture plate, 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td>Support</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM (Dulbecco&#39;s Modified Eagle&#39;s Medium- high glucose)</td>
			<td>Sigma</td>
			<td>D5671</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>ED4SS-500g</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Epi Episomal iPSC Reprogramming Kit</td>
			<td>Invitrogen</td>
			<td>A15960</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FAST - READ 102</td>
			<td>Biosigma</td>
			<td>BVS100</td>
			<td>Tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin</td>
			<td>Merck</td>
			<td>FC010</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>Reagent</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">H2O</td>
			<td></td>
			<td>MILLIQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Thermofisher</td>
			<td>33342</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminin 521</td>
			<td>Stem Cell Technologies</td>
			<td>77003</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine (200 mM)</td>
			<td>Gibco</td>
			<td>LS25030081</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrigel</td>
			<td>Corning Matrigel hESC-Qualified Matrix</td>
			<td>354277</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:589px;">Mouse embryonic fibroblasts (MEF)</td>
			<td>Life Technologies</td>
			<td>A24903</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Medium</td>
			<td>Stem Cell Technologies</td>
			<td>85851</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MTESR1 Supplement</td>
			<td>Stem Cell Technologies</td>
			<td>85852</td>
			<td>Medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phalloidin</td>
			<td>Sigma</td>
			<td>P1951</td>
			<td>Reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vitronectin</td>
			<td>Stem Cell Technologies</td>
			<td>7180</td>
			<td>Coating</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td>Reagent</td>
		</tr>
	</tbody>
</table>

measuring the confluence of ipscs using an automated imaging system

이 연구는 다양한 ECM 코팅 기판에서 성장하는 iPSC가 세포 합류에 어떤 영향을 미칠 수 있는지 이해하는 데 중점을 둡니다. 성장 섭동을 피하기 위해 단일 세포 현탁액의 세포를 계산할 필요 없이 실시간으로 iPSC 합류를 평가하는 프로토콜이 확립되었습니다. 고함량 이미지 분석 시스템을 사용하여 시간이 지남에 따라 4개의 서로 다른 ECM에서 iPCS 합류를 자동화된 방식으로 평가했습니다. 부착성 iPSC의 세포 합류를 평가하기 위해 다양한 분석 설정을 사용했으며 60, 80 또는 100% 마스크가 적용되었는지 여부에 관계없이 약간의 차이(라미닌의 경우 24시간 및 48시간)만 관찰되었습니다. 우리는 또한 라미닌이 Matrigel, vitronectin 및 fibronectin에 비해 가장 좋은 합류로 이어진다는 것을 보여줍니다.

이 연구에서는 다양한 코팅 조건에서 성장할 때 iPSC의 합류를 조사했습니다. 세포 분석기를 사용하여 5 일 만에 3 배로 쉽게 유익한 결과를 얻을 수있었습니다. iPSC는 플라스틱 용기에 거의 부착되지 않고 증식을 지원하기 위해 코팅이 필요하기 때문에 세포 배양의 건강을 나타내고 분화 가능성을 반영할 수 있으므로 인간 iPSC의 합류를 모니터링하기로 결정했습니다. 시험관 내 확장 후, 우리는 iPSC...

Watch this Scientific Journal Video about Measuring the Confluence of iPSCs Using an Automated Imaging System at JoVE.com

Measuring the Confluence of iPSCs Using an Automated Imaging System

이 프로토콜의 목표는 다양한 세포외 기질(ECM) 코팅 조건을 비교하여 차등 코팅이 유도만능줄기세포(iPSC)의 성장 속도에 어떤 영향을 미치는지 평가하는 것입니다. 특히, 우리는 iPSC 배양의 최적 성장을 얻기위한 조건을 설정하는 것을 목표로합니다.

자동 이미징 시스템을 사용한 iPSC의 합류점 측정.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...

measuring-the-confluence-of-ipscs-using-an-automated-imaging-system

Research

JoVE Journal

Biology

1.9K 조회수.  Ospedale Pediatrico Bambino Gesù, IRCCS. 이 프로토콜의 목표는 다양한 세포외 기질(ECM) 코팅 조건을 비교하여 차등 코팅이 유도만능줄기세포(iPSC)의 성장 속도에 어떤 영향을 미치는지 평가하는 것입니다. 특히, 우리는 iPSC 배양의 최적 성장을 얻기위한 조건을 설정하는 것을 목표로합니다.

비디오: Measuring the Confluence of iPSCs Using an Automated Imaging System

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

절단 수술을받은 사람에 대한 Osseointegrated 지능형 임플란트 설계 시스템의 생체 분석

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

연구

생물학

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

광학 트랩을 사용하여 박테리아 세포의 굽힘 강성을 측정

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

이미징을위한 간단한 방법 Arabidopsis는 Infiltrative 영상 매체로 Perfluorodecalin를 사용하여 잎

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.

The use of a 3D automatic video system that can track individual and groups of zebrafish is described. As application example we explore the effects of the NMDA-receptor antagonist MK-801 on shoals of zebrafish.

기록 개인과 성인 Zebrafish의 떼에 자동 3D 추적 시스템을 사용하여

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

에 박테리아의 효과를 측정<em&gt; C. 엘레</em계란 보존 분석을 사용하여&gt; 행동

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

담배 과도 식 시스템을 사용하여 MAPK 신호 전달 통로의 공간 개편 이미징

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

단백질의 품질 관리 시스템을 공부<em&gt; D. discoideum</em&gt; 온도 제어 라이브 셀 이미징을 사용하여

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

마이 오신 중쇄 면역를 사용하여 쥐의 근육 크로스 섹션에서 근육 섬유 인구 분석을위한 신속한 자동화 프로토콜

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

자동된 경도 Luciferase 가스 온도 최적화 레코더 (악어) 이미지를 사용 하 여 발광 기자 들의 유연한 측정

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...