우리는 37 ° C PFA에 고정을 시도하는 제안을 포함하여 도움이 토론 박사 해롤드 드보락을 인정합니다. 이 작품은 NIH 보조금 P01의 CA92644에 의해 암 연구를위한 국립 재단의 계약에 의해 지원되었다.

1. 콜라겐 코팅 유리 디스크에 세포 배양 <ol><li> 유리 항아리 (4온스) 및 디스크를 살균하는 오토 클레이브에 넣고 유리 디스크 (직경 12mm). </li><li> 50 ML 팔콘 튜브에 25 ㎖의 70 % 에탄올을 넣고 세포 배양 후드에 배치합니다. 용액의 레벨이 20 ㎖로 떨어질 때까지이 솔루션은 여러 번 재사용 될 수있다. </li><li> 유리 디스크를 처리 할 수​​ 있도록 사용하기 전에 5 분 동안 70 % 에탄올에 엑스트라 파인 지점과 날카로운 집게를 놓습니다. </li><li> 조심스럽게 튜브의 집게를 제거하고 뚜껑을 부드럽게 2 분 동안 건조한 공기 튜브의 상단에 에탄올 처리 집게를 배치합니다. 집게의 끝이 후드에 아무것도 만지지 않도록하십시오. </li><li> 유리 항아리 하나 멸균 유리 디스크를 잡아 24 웰 세포 배양 접시의 우물에 배치하는 멸균 집게를 사용합니다. 우물의 원하는 번호가 디스크로 채워 될 때까지 반복합니다. </li><li> 소 콜라겐 용액 500 μl를 (넣어50 NG / ML) 유리 디스크를 포함하고 적어도 30 분 동안 37 ° C, 5 % CO 2 세포 배양 인큐베이터에 배치 각 웰에. 더 이상 배양에 해가 없습니다. </li><li> 수확 HUVEC (사람의 제대 정맥 내피 세포) 또는 PC3 이미 트립신 통해 150mm 세포 배양 접시에서 성장 및 그 완전 배지를 사용하여 트립신 활성을 차단 하였다 (전립선 종양 세포). 15 ML 팔콘 튜브에 세포를 수집합니다. </li><li> 세포 수를 계산하고 생존 능력이 90 % 이상 있는지 확인합니다. </li><li> 4 ℃에서 10 분 동안 200 XG에서 원심 분리하여 세포 펠렛 뜨는을 제거합니다. </li><li> 10 105 세포 / ㎖로 희석하여 세포 배양 배지를 사용한다. 얼음에 세포를 놓습니다. </li><li> 세포 배양 후드에 콜라겐을 대기음 부드럽게 유리 디스크가 콜라겐 예비 코팅 된 잘 각각의 세포 현탁액 500 μl를 놓습니다. 24 - 웰 플레이트에 5 × 104 세포 / 웰의 60 %에게 컨퍼런스을 줄 것이다HUVEC과 PC3 세포에 대한 30 %의 포화 상태에 대한 luency. 참고 : 높은 세포 밀도에 대한 더 많은 세포 현탁액을 추가합니다. </li><li> 문화 37 ° C, 1 시간, 2 시간, 4 시간, 6 시간, 또는 nanopodia 활동과 세포 통로를 추적하는 24 시간 동안 5 % CO 2 배양기에서 세포. 전형적으로, 세포는 처음 30 분에 유리 디스크에 부착하고, 그 후에 편광 및 제 시간에 마이그레이션 시작하고, 다음 시간에 세포 간 상호 작용 및 세포 분열을 수행한다. </li></ol> 2. 셀 고정 <ol><li> 각 웰은 세포를 해결하기 위해 500 ㎕의 4 % PFA (파라 포름 알데히드)가 필요합니다. 상업적으로 제조 또는 갓 4 % PFA 사용할 수 있습니다 만든 하나. 제조 웰의 수에 기초하여 필요한 PFA의 양을 계산하고, 15 ㎖의 팔콘 튜브에 PFA를 배치. 주의 : 파라 포름 알데히드는 발암 물질로 의심입니다. </li><li> 37 ° C의 배양기에서 장소에 이미 스티로폼 스탠드 팔콘 튜브를 넣어. 전쟁 30 분 동안 인큐베이터에서 튜브 남기기37 ° C.에 PFA를 해요 </li><li> 문화 후드에 전기 패드를 배치하고 전원을 켜십시오. </li><li> 24 웰 세포 배양 플레이트와 인큐베이터 밖으로 데워진 PFA를 꺼내 가열 패드에 올려 놓습니다. </li><li> 우물에서 매체를 대기음 한 손을 사용 후 즉시 부드럽게 잘 가장자리를 통해 우물에 500 ㎕의 PFA를 슬라이드에 다른 손을 사용합니다. 쉽게 흡입의 경우, 그 각도에서 안정되도록 스티로폼 스탠드에 기대어, 약 45 ℃에서 배양 접시를 기울이십시오. 이는 흡입 및 배양 세포를 방해하지 않고, 우물 PFA 용액을 첨가를 촉진 할 것이다. 이것은 세포 활동의 일본어 셀룰러 구조를 보존하기 위해 가장 중요한 단계이다. </li><li> 유리 디스크를 모두 우물 PFA를받을 때까지이 과정을 반복합니다. 참고 : 우물에서 기존 솔루션을 변경하는 데 필요한 모든 단계를 같은 포부 절차를 적용합니다. </li><li> 5 분 동안 37 ° C에서 접시를 놓습니다. </li&gt; <li> 다시 문화 후드에 접시를 가지고 단계 2.5에 설명 된대로 스티로폼에 기울이십시오. 부드럽게 수집 관으로 우물에서 PFA를 전송 한 손을 사용하여; 이후에 즉시 잘 적어도 500 ㎕의 방 온도 PBS를 배치 할 다른 손을 사용합니다. 모든 우물 세척 한 후, 돌아가서 잘 때마다 한 번 더 PBS 세척을 반복합니다. 유해 폐기물 용기에 수집 된 PFA를 비 웁니다. </li><li> PBS로 2 % 소 태아 혈청을 추가하여 ICC 차단 버퍼를 준비합니다. 0.04 % 소듐 아 지드 (20 % 원액으로부터 희석) 방부제로서 첨가된다. 주의 : 아 지드 화 나트륨은 독성 화합물이다. 버퍼는 세균 오염없이 장기간 동안 39 ° C에서 저장 될 수있다. </li><li> 여전히 서로 잘 PBS를 제거하는 흡입하고 그 후 즉시 ICC 차단 버퍼 500 μl를 추가하는 순환 다시 한 번, 스탠드를 기울이면 배양 접시. 세포는 이제 면역 형광 염색을위한 준비가되어 있습니다. 필요한 경우,고정 된 세포는 TM4SF1 단백질의 상당한 손실없이 일주일 동안 4 ° C의 냉장고에 저장 될 수 있습니다. </li></ol> 3. Nanopodia의 TM4SF1 면역 형광 염색법 <ol><li> 각 웰에서 트리톤 X-100, 0.01 %를 함유하는 새로운 블로킹 완충액 500 μL의 ICC 블로킹 버퍼를 교체하고 실온에서 1 시간 동안 앉아 보자. 이 세포막에서 TM4SF1가 제거됩니다로 0.03 % 트리톤 X-100보다 더 높은 사용하지 마십시오. 염색은 바로 시작할 준비 경우 PBS 단계 2.10에서 세포를 세정 직접 트리톤을 함유하는 블로킹 버퍼에 이동 될 수있다. </li><li> ICC 차단 버퍼에 0.5 ㎍ / ㎖의 1 차 항 TM4SF1 (또는 항-CD9) 항체를 희석. </li><li> 각 웰에 ICC/0.01 % 트리톤 버퍼를 제거하고 방지 TM4SF1 - 항체 용액 300 μL로 교체합니다. 실온에서 2 시간을 품어 또는 4 ℃에서 하룻밤 남겨 </li><li> 더 적은 500보다 μL PBS로 각 잘 씻으십시오. 3 회 반복; 마다 르에 제공배양 시간의 AST 5 분. </li><li> 에있는 이차 항체와 phalloidin도 솔루션을 준비 ICC 알렉사 488 표지 당나귀 항 마우스 이차 항체 (2 ㎍ / ㎖의 최종 농도)의 1000 배 희석 및 phalloidin도 1000 X 희석 (50 NG / ML 최종 농도)를 사용하여 버퍼를 차단. </li><li> PBS를 제거하고 각 웰에 차 항체 및 phalloidin도 용액 300 μl를 추가하고 2 시간 동안 배양 또는 4 ℃에서 하룻밤 남겨 </li><li> 세척 당 적어도 5 분의 배양과 PBS 세척 단계를 반복합니다. 마지막 세척에서 1 시간 동안 PBS에서 잘 각을 둡니다. </li><li> 유리 슬라이드에 방지 페이드 설치 미디어의 한 방울 (~ 10 μl를) 삭제합니다. </li><li> 벤드 하드 표면에 부드럽게 눌러 주사기 바늘 90 °에서 19 G 1 ½의 끝. 굽​​은 바늘을 잡고 조심스럽게 우물에서 유리 디스크를 들어 올린 날카로운 집게를 사용하여 디스크를 파악하기 위해 다른 손을 사용하여 한 손을 사용합니다. </li><li> 유리 디스크 및 회전# 160; 유리 슬라이드에 세포 측 콘택트 설치 미디어를주는 얼굴을 아래로. 부드럽게 유리 디스크를 배치하고 상온에서 어두운 곳에서 하룻밤 건조 할 수 있습니다. </li><li> 이미지에 대한 면역 형광 염색 nanopodia를 진행, 또는 -20 ℃에서 슬라이드 상자에 슬라이드를 저장 슬라이드는 -20 ° C에서 몇 달 동안 볼 수 유지됩니다 </li></ol>

<ol>
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관심 없음 충돌 선언하지 않습니다.

Nanopodia 단단히 TMED을 통해 매트릭스에 부착 환경을 감지하고 세포 간 상호 작용 2,3을 중재 할 수있는 편광 모바일 휴대에서 100 개 이상의 μM을 확장 할 수 있습니다 얇은 세포막 채널입니다. 그래서 제대로 잔기가 멀리 이동함에 따라 셀 남아 있는지 매트릭 밀착 Nanopodia (도 1 및 2). Nanopodia 따라서 우리는 세포가 자신의 환경을 감지하는 방법을 연구하고 세포의 이...

운동을위한 편광 동안, 동물 세포는 filopodia, lamellipodia는 후퇴 섬유, 프릴 1 포함한 표면에서 동적, 막 돌기의 다양한 확장합니다. 최근이 목록에 추가 nanopodia, 얇은의 새로 인식 형 (직경 100 ~ 300 μm의), 같은 filopodia 같은 F-액틴 구조의 확장을위한 막 채널을 제공 연장 (최대 50 ~ 100 μm의 길이) 막 투영했다 과 후퇴 섬유, 그리고 얼룩 긍정적 TM4SF1 (횡단-4-L-여섯 가족 1) 배양 된 내피 세포와 종양 세포 2,3에와. TM4SF1 원래 분자가 내피 세포의 증식과 이주 2,3에서 필수적인 역할을 내피 세포 바이오 마커이다 발견하기 전에 종양 세포 항원 4 알려진 tetraspanin 같은 토폴로지 단백질이다. 면역 형광 염색 TM4SF1가 perinuc하는 지역화 된 것으로 나타났다리어 소포와 세포막에, 그리고는 TM 4SF1 전자 nriched 마이크로 D omains (TMED)에 충실. TMED 앵커 매트릭스 nanopodia 및 nanopodia 1-3 TMED / μm의 길이의 정기적 간격 줄무늬 패턴에 존재. Nanopodia는 일반적으로 세포의 주요 전방에서 연장 운동을위한 세포 편광 동안 뒤 후행. 때문에 TMED의 회사 부착 특성으로 nanopodia는 멀리 이동할 때 셀에 다시 철회 할 수 없습니다; 버려진 nanopodia 잔기 따라서 셀룰러 이동 경로를 추적. F-액틴 확장과 후퇴 막 채널을 제공하고, 세포 간 상호 작용 및 통신 2,3의 사이트는 Nanopodia. 이러한 특성은 세포의 양극화, 환경의 휴대 감지, 세포 운동의 경로와 방향을 결정하고, 세포 간 상호 작용을하는 동안 F-액틴 어셈블리를 기본 메커니즘을 연구 할 수있는 독특한 기회를 제공 nanopodia 의미차 통신. 때문에 TMED의 소수성이 강한 성격과 nanopodia의 얇고 깨지기 쉬운 막 자연에 특별한주의는 TMED 및 nanopodia을 유지하기 위해 수행해야합니다. nanopodia 및 일반 실험실 방법으로 TMED 제거의 파괴는 예순셋 출판물은 1986 년에 처음 발견 이후와에 100 ~ 300 μm의 마이크로 도메인에서 TM4SF1 농축 지식의 완전한 부족 TM4SF1에 출연 한 이유 가능한 이유 세포 표면 2009/05/02에있는 내피 세포에서 TM4SF1의 보고서까지. 기존의 면역 염색 방법은 일반적으로 세포를 고정하고 세포에게 5 투과 0.1 % 이상 트리톤 X-100의 농도를 사용하는 에탄올, 메탄올, 아세톤과 같은 유기 용매를 사용합니다. (내가) 37 ° C 4 % PFA를 사용하고 37 세포를 수정 : 여기에 설명 된 연구는 TMED 및 nanopodia가 공개하는 종래의 방법으로 세 가지 주요 변경 사항을 구현76, C 인큐베이터, (II) 부드러운 실내 온도 PBS 세척을 적용, 그리고 (iii) 트리톤 X-100은 트리톤 X-100 0.03 %보다 높은 추출하므로, 일차 항체를 첨가하기 전에 세포를 투과하기 위해 잠깐 동안 만 0.03 % 이하를 사용 TM4SF1. 모든 tetraspanins는 세포막 06 마이크로 도메인을 형성하고 일부는 내피 세포 및 종양 세포에서 2,3 TMED로 colocalize. CD9, CD81, CD151과 같은 tetraspanins이 재하 표현되는 바와 같이, 여기에 기재된 염색 프로토콜 nanopodia 기능 연구에 TM4SF1 부족 많은 다른 세포 유형으로 확장 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="1597">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass discs&#160;</td>
			<td style="width:160px;">Fisher Scientific</td>
			<td style="width:133px;">12-545-82&#160;</td>
			<td style="width:983px;">12-mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass jar</td>
			<td>Fisher Scientific</td>
			<td>02-912-310</td>
			<td>Certified Clean Clear Glass Straight-Sided Jars, 4 oz.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass slide</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">12-544-1</td>
			<td>Fisherfinest Premium Plain Glass Microscope Slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50-ml falcon tube&#160;</td>
			<td style="width:160px;">BD Falcon</td>
			<td style="width:133px;">352070</td>
			<td>50-ml high-clarity polypropylene conical centrifuge tube, 16,000 rcf rating. Sterile.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">15-ml falcon tube with Styrofoam rack</td>
			<td style="width:160px;">Corning</td>
			<td style="width:133px;">430790</td>
			<td>Corning 15-ml PP Centrifuge Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sharp forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">13-812-42</td>
			<td>Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">24-well cell culture plate</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353047</td>
			<td>24-well Cell Culture Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">150-mm cell culture plate&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">353025</td>
			<td>150-mm cell culture plate&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">19G 1 &#189; syringe needle&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">309635</td>
			<td>19G 1 &#189;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Reagent</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ethanol</td>
			<td>Decon Laboratories, Inc.</td>
			<td>2701</td>
			<td>Decon&#39;s Pure Ethanol 200 Proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">collagen solution&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">354249</td>
			<td>Collagen I, High Concentration, rat tail, 100 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">trypsin/EDTA</td>
			<td style="width:160px;">Cellgro</td>
			<td style="width:133px;">25-053-CI</td>
			<td><a name="RANGE!D15">0.25% Trypsin-EDTA 1X</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">75889 5 LT</td>
			<td style="width:983px;">PBS, 10X Solution, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PFA (paraformaldehyde)</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">19943 1 LT</td>
			<td>4% in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500ML</td>
			<td><a name="RANGE!D19">Fetal Bovine Serum</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">EGM2-MV</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>EGM-2 BulletKit, EBM-2 Basal Medium 500 ml and EGM-2 SingleQuot Kit Suppl. &#38; Growth Factors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">T8787</td>
			<td>Triton X-100</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaN3</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">S2002-25G</td>
			<td>solubilized in water. 4% stock solution and working concentration is 0.4%.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human TM4SF1 antibody&#160;</td>
			<td style="width:160px;">Millipore</td>
			<td style="width:133px;">MAB3127</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human CD9 antibody&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">555370</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Alexa Fluor 488 Donkey anti-mouse 2nd antibody</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">A-21202</td>
			<td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Phalloidin</td>
			<td style="width:160px;">Chemicon</td>
			<td style="width:133px;">90324</td>
			<td>Rhodamine-conjugated Phalloidin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">anti-fade mounting media&#160;</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">P-36931</td>
			<td>ProLong Gold Antifade Reagent with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Buffers and solutions</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">70% ethanol&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td>17.5 ml of ethanol (200 proof) 
			7.5 ml of double deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS (make 200 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20 ml of 10X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">180 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20% Triton X-100 (make 20 ml)&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">4 ml</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">16 ml double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">4% NaN3 (make 25 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">1 g of NaN3</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">50 ng/ml bovine collagen solution in PBS (make 5 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Make stock soln. of 50 &#956;g/ml (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">830 &#956;l of Collage I (3 mg)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml PBS&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ICC Blocking Buffer (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">49 ml PBS</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2% FBS (add 1 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">0.04% NaN3 (add 100 &#956;l of 4% NaN3)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml of ICC Blocking Buffer&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 &#956;l of 20% Triton X-100</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Cell</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HUVEC&#160;</td>
			<td style="width:160px;">Lonza&#160;</td>
			<td>C2517A</td>
			<td><a href="http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx">http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PC3</td>
			<td style="width:160px;">ATCC</td>
			<td style="width:133px;">CRL-1435</td>
			<td><a href="http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7">http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Equipment</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">cell culture hood</td>
			<td style="width:160px;">NuAIRE</td>
			<td style="width:133px;">Nu-425-600</td>
			<td>NU-425 (Series 60) BIOLOGICAL SAFETY CABINET&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">37 &#176;C, 5% CO2 cell culture incubator&#160;</td>
			<td style="width:160px;">CellStar</td>
			<td style="width:133px;">QWJ300DABA&#160;</td>
			<td>Cellstar CO2 Water Jacketed Incubator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">heating pad&#160;</td>
			<td>K&#38;H Manufacturing</td>
			<td style="width:133px;">1020</td>
			<td style="width:983px;">K&#38;H Lectro Kennel Heated Pad with Free Fleece Cover (http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5x28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">centrifuge</td>
			<td style="width:160px;">Sorvall&#160;</td>
			<td style="width:133px;">T6000B&#160;</td>
			<td>Sorvall T6000 (B) Benchtop Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">Z359629</td>
			<td>Bright-Line Hemacytometer</td>
		</tr>
	</tbody>
</table>

nanopodia - thin, fragile membrane projections with roles in cell movement and intercellular interactions

문화에 부착 세포 이동 및 세포 간 상호 작용을 지원하기 위해 편광 상태를 유지한다. Nanopodia는, 얇은 긴, TM4SF1 (횡단-4-L-여섯 가족 1) 면역 형광 염색을 통해 시각화 할 수있는 내피 세포와 암 세포에서 주로 F-액틴 음성 막 전망이다. 100 ~ 300 μm의 직경 TMED에서 TM4SF1 클러스터 많은 14 개별 TM4SF1 분자에 3을 포함 (TM 4SF1 전자가 마이크로 D omains을 nriched). TMED는 1 μ의 m 당 TMED 3 단단히 행렬에 nanopodia 앵커의 일정한 간격으로 nanopodia 따라 간헐적으로 배치되어있다. 이 편광 내피 또는 종양 세포의 전면을 선행 또는 후행 후면에서 100 개 이상의 μ m를 확장 할 nanopodia을 가능하게하고, MATR에 남아 수 막 잔류의 원인이IX 세포가 멀리 이동하면. TMED 및 nanopodia 때문에 자신의 극단적 인 취약성 및 온도에 대한 민감도 간과되었다. 일상적인 세척 및 고정이 구조를 방해. Nanopodia는 염색 전에 0.01 % 트리톤 X-100에 대한 간단한 노출 한 후, 37 ° C에서 파​​라 포름 알데히드 (PFA)에 직접 고정하여 유지됩니다. 세포 생물학의 새로운 풍경을 열어 Nanopodia : 그들은 감지하고 거리에서 다른 세포를 식별, 가까운 접촉에서 세포 간 상호 작용을 시작하고, 운동 확산에 관련된 신호 전달 메커니즘, 세포가 자신의 환경을 감지하는 방법에 대한 우리의 이해를 바꿀, 세포 약속 세포 통신. TM4SF1 파생 nanopodia을 연구 개발하는 방법은 고전적인 tetraspanins, 특히 재하 표현 CD9, CD81, 및 CD151의 기관을 통해 다른 세포 유형에 형성 nanopodia의 연구에 유용 할 수 있습니다.

1 단계의 경우 : (예 : HUVEC과 본 연구에서 사용 된 PC3 등) 세포가 정상적으로 성장 할 수있는 경우, 세포가 씨를 뿌린 후 30 분 안에 콜라겐 코팅 된 디스크에 부착 편광 곧 이후에 이동되고, 앞으로의 경로 nanopodia을 확장 할 것입니다 운동.도 1a 및도 1b는 각각 편광 및 증식 HUVEC.도 2a는 이동 상태에서 편광 PC3 세포를 도시 나타낸다. 2 단...

Watch this Scientific Journal Video about Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions at JoVE.com

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Nanopodia 셀의 전면을 선행 또는 후행 후면에서 100 μM까지 확장하고 세포 환경을 감지 얇고 깨지기 쉬운 막 채널입니다. 37 ° C에서, 부드러운 세정, 에탄올, 메탄올, 아세톤과 같은 유기 용매의 방지에 높은 트리톤 X-100 농도의 직접 고정 이러한 세포 구조를 관찰해야합니다.

Nanopodia - 세포 이동 및 세포 간 상호 작용의 역할로 얇고 깨지기 쉬운 막 계획

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

nanopodia-thin-fragile-membrane-projections-with-roles-cell-movement

Research

JoVE Journal

Biology

10.2K 조회수.  Harvard Medical School.  Nanopodia 셀의 전면을 선행 또는 후행 후면에서 100 μM까지 확장하고 세포 환경을 감지 얇고 깨지기 쉬운 막 채널입니다. 37 ° C에서, 부드러운 세정, 에탄올, 메탄올, 아세톤과 같은 유기 용매의 방지에 높은 트리톤 X-100 농도의 직접 고정 이러한 세포 구조를 관찰해야합니다. 

비디오: Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

루시페라제 기반 리포터 유전자 분석과 함께 막 Biogenesis 유학

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

연구

생물학

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL에서 MultiBac 단백질 복잡한 생산 플랫폼

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

CA의 역할을 평가하기위한 영상 분석을 라이브<sup&gt; 2 +</sup기공 형성 독소 상처의 수리에&gt;와 Sphingomyelinase

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

갭 접합부 세포 간 통신 : 천연 제품의 독성 물질과 독소의 반작용을 평가하기위한 기능성 바이오 마커, 보건 혜택

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

VirusMapper 슈퍼 해상도 현미경을위한 오픈 소스 단일 입자 분석

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

플래시 및 동결 : 전자 현미경으로 막 역학을 캡처하는 소설 기법

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

거 대 한 Unilamellar 소포에서 막 나노튜브를 당기

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

FRET-Sensitized Emission with FRET-Sensitized Release를 사용한 살아있는 세포의 단백질 상호 작용 평가

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

지질 막 역학 및 막 단백질 상호 작용을 위한 독특한 프로브로 중성자 스핀 에코 분광법

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

전기 생리 학적 연구를위한 성체, 청소년, 애벌레 및 배아 제브라 피쉬에서 심장 및 혈관 평활근 세포 분리

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...