우리는 이러한 프로토콜의 발전에 기여 Gelfand는 실험실의 구성원을 감사드립니다. 이 책에서보고 된 연구는 보너스 번호 BFI-2011-295 UDC에 아래 VIG에 보너스 번호 R01GM052111에서 건강의 국립 연구소의 일반 의료 과학 국립 연구소 바스크 정부 교육부, 대학 및 연구에 의해 지원되었다.

1. 초파리 S2 세포 <ol><li> CONA 코팅 커버 슬립의 제조 <ol><li> 세라믹 랙에 커버 슬립을 놓고 1 시간 동안 크롬산 침지을 씻어. [주의 : 크롬산은 부식성이 눈의 자극, 코, 목, 피부가 발생할 수 있습니다]. </li><li> 산을 완전히 세척 될 때까지 지속적으로 30 분 동안 dH보다 2 O를 실행 충분히 커버 슬립을 씻어. </li><li> 하자의 coverslips 공기 건조하고 CONA 30 분 DH (2 O 0.5 ㎎ / ㎖ 용액)와 코트를. </li><li> 15 분 dH보다 2 O와 커버 슬립을 씻어하고 그들에게 자연 건조시켜. CONA 코팅 coverslips는 1 개월까지 저장할 수 있습니다. </li></ol></li><li> 세포 도금 <ol><li> S2 세포는 지수 적 실험에 필요한 세포의 수에 따라 T25 또는 T75의 cm 2 플라스크에서 성장하는 것을 허용한다. </li><li> 혈구를 이용하여 세포 밀도를 계산. 추가 1 ㎖ 성장 M부드럽게 35mm 조직 문화 CONA 코팅 coverslip에와 요리, 그리고로 edium (예를 들어 곤충-익스프레스)는 요리에 ~ 1 × 10 5 세포를 전송합니다. 서로 다른 셀로부터의 프로세스들이 중첩되지 않기 때문에이 세포 밀도는 촬상에 적합하다. </li><li> 즉시 (2.5 μM C​​ytoD의 최종 농도) 접시에 5 μM의 CytoD으로 1 ㎖ 매체를 추가 도금 후, 프로세스의 형성을 유도하기 위해. 촬영 전에 25 º C에서 최소 2 시간 동안 세포를 배양하여 프로세스의 전체 개발을 허용합니다. </li></ol></li></ol> 2. 초파리 차 신경 세포 문화 <ol><li> 신경 세포 배양을위한 준비 <ol><li> (:, 5 ㎍ / ml의 인슐린, 100 ㎍ / ㎖의 페니실린, 100 ㎍ / ml의 스트렙토 마이신, 10 ㎍ / ㎖의 테트라 사이클린 20 % 소 태아 혈청, 30 분 동안 55 º C에서 불 활성화 열 슈나이더의 매체로 보충) 초파리 신경 세포의 성장 매체를 준비합니다 . 이 SUPplemented 슈나이더의 매체는 6 개월 4 º C에 저장할 수 있습니다. </li><li> Drosophila의 배아 (22.5 g 한천 12.0 g 자당 750 ML의 H 2 리터 플라스크에 2 O를 수집하기 위해 사과 주스 한천 유리 병을 준비, 20 ~ 25 분 동안 오토 클레이브는, 250 ml의 사과 주스를 추가하고 각 초파리에 ~ 10 ㎖를 부어 유리 병;) 응고 후,면 공 튜브를 연결하고 식품의 건조를 통해 방지하기 위해 플라스틱 음식 포장으로 유리 병 포장. 한천 병에 사과 주스는 4 주 동안 잘 밀봉 된 비닐 봉투에 4 º C에 저장할 수 있습니다. </li><li> CONA와 코트 산 세척 25mm 원형 커버 슬립 (초파리 S2 세포에 대한 프로토콜 참조)를 10 ~ 15 분 동안 자외선 아래를 소독. 이러한 커버 슬립 개월 동안 저장 될 수있다. </li></ol></li><li> Drosophila의 배아 신경 세포의 배양 <ol><li> 계속 성인 배아 F를 수집하기 전에 1-2일에 건조 효모 보충 사과 주스 한천 병 난다또는 신경 준비. </li><li> 수집 일에, 수집 된 배아를 동기화하기 위해, 전송 성인 빛 1 시간 precollection에 대한 신선한 사과 주스 한천 유리 병에 날아 이러한 배아를 폐기합니다. </li><li> 전송은 1 시간 동안 배아를 수집하고, 배아의 수율을 높이기 위해 어둠 속에서 모두 다른 시간에 다른 유리 병에 파리를 전송하고, 새로운 사과 주스 한천 유리 병에 파리. </li><li> 사과 주스 한천 유리 병에서 파리 성충을 제거하고 배아 실내 온도 (4 ~ 6 시​​간짜리 배아 단계 9 ~ 11 사이)의 단계에 또 다른 4 시간 9 ~ 11 호를 개발하자. </li><li> 유리 병에 물을 분출하고 (최소 10 배아는 하나의 좋은 신경 세포의 준비에 필요한) 작은 붓 사과 주스 한천의 배아를 풀어 배아를 제거합니다. </li><li> 물을 배수 할 수있는 전송 피펫으로 100 μm의 나일론 메쉬 셀 스트레이너에 배아를 전송 (Drosophila의 배아 세척 버퍼, 0.4 % 염화나트륨, 0.03 % 트리톤의 전송 피펫을 사전 헹굼X-100). </li><li> 작은 붓을 사용하여 여과기에서 배아를 수집하고, 1 ㎖ Drosophila의 배아 세척 (참고 : 페인트 브러시 또는 피펫 팁을 사용하는 한천 또는면 섬유를 청소)로 채워진 1.5 ml의 마이크로 튜브로 전송할 수 있습니다. 이 프로토콜에서, 1.5 ml의 마이크로 튜브는 일치하는 처분 할 수있는 펠릿 유와 함께 제공됩니다. </li><li> Drosophila의 배아 세척 배에서 배아를 씻으십시오. </li><li> 10 분 동안 신선한 1시 1분 상업 표백제 용액 및 95 % 에탄올에 배아를 Dechorionate [주의 : 표백제 자극 눈, 코, 목, 피부가 발생할 수 있습니다]. </li><li> 조직 문화 후드로 이동 한 보충 슈나이더의 매체에 배아 배를 세척한다. </li><li> 배아와 튜브에 200 ~ 300 ㎕의 매체를두고 부드럽게 기계 (10-15X를 갈기) 70 % 에탄올로 소독 한 킴블 체이스 처분 할 수있는 펠릿 방앗 공이를 사용하여 dechorionated 배아를 떼어 놓다. </li><li> 2 분, 그리고 TRA 20 XG에 균질 혼합물을 원심 분리기새로운 튜브에 뜨는 nsfer. [이 단계는 선택 사항입니다, 그것은 배아 파편을 제거하는 데 도움이됩니다] </li><li> 세포 펠렛 2 분 550 XG에 뜨는을 원심 분리기. </li><li> 2 분 550 XG에 다시 슈나이더의 중간 스핀 세포를 보충 500 μL에 펠렛을 재현 탁, 한 번 재 부유 - 회전주기를 반복합니다. </li><li> 마지막으로 슈나이더의 매체를 보충 ~ 100 μL에 펠렛을 재현 탁하고 멸균 35mm 페트리 접시에 25mm CONA 코팅 커버 슬립에 세포를 접시. 세포 부착을 허용하기 위해 10 분 동안 배양한다. </li><li> 부착 된 세포와 커버 슬립을 잠수함 5 μM C​​ytoD 보충 슈나이더의 배지 2 ㎖를 추가합니다. </li><li> 촬영하기 전에 밤새 가습 25 º C의 배양기에서 접시를 품어. </li></ol></li></ol>

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저자는 더 경쟁 재정적 이익이 없다는 것을 선언합니다.

여기에서 우리는 초파리 S2 세포와 차 신경 세포 배양에서 미세 소관 따라화물 수송 공부를위한 상세한 프로토콜을 제시한다. 두 S2 프로세스와 신경 돌기는 세포 기관의 수송을위한 트랙 역할을 번들로 미세 소관 배열로 채워 구조입니다. 두 가지 전략은 일반적으로 라벨 세포 기관에 사용됩니다 문화에 직접 추가 형광 염료를 가진 세포 기관 타겟팅 순서 나 염색과 ?...

초파리 S2 세포는 많은 세포 과정을 공부 증가 인기를 얻고있다. 이러한 세포는 원래 OregonR 초파리 melanogaster의 트립신의 최종 단계의 배아로부터 얻어지는 1. 초파리 S2 세포가 다른 세포주에 비해 많은 장점을 제공 식세포와 같은 기능을 유지 하였다. 그들은 CO 2없이 25 ° C에서 배양하고, 가열 또는 가스 교환의 필요없이 상온에서 많은 시간 동안 몇 군데하실 수 있습니다. 더 중요한 것은, 초파리 S2 세포는 그 단백질의 고효율 최저를 허용 RNAi의 치료에 매우 민감하다. S2 세포는 높은 형질이며 안정적인 세포주는 또한 단백질의 안정 이소성 발현을 허용 미만 사주에서 선택 될 수있다. S2 세포는 일반적으로 하나 성장 느슨하게 연결된하지만 플라스크 또는 정지에 확산되지. 그들은 식물 렉틴 콩카와 프리 코트 커버 슬립에 확산하도록 유도 할 수있다navalin A (CONA). 확산 세포의 주변은 특히 얇은 때문에 살아있는 세포 현미경에 이상적입니다. S2 세포 배양, RNAi의 치료, 라이브 광 이미징 및 면역 염색에 대한 자세한 프로토콜은 문학 2,3에서 찾을 수 있습니다. 우리 연구실은. 같은 사이토 D로 F-액틴을 해중합 또는 끊고 마약, (CytoD)으로 처리 초파리 S2 세포가 균일하게 편광 미세 소관 4 가득 긴 프로세스를 성장 미세 소관 기반의화물 운송을 연구하는 모델 시스템으로 S2 세포를 채택했다 10, 그 미세 소관 배열에 따라화물의 움직임을 연구 할 수있는 이상적인 시스템을 만드는. 이러한 과정에서 전송의 다른 매개 변수 (. 즉 궤도의 길이, 속도, 방향성 등)을 쉽게 자동화 된 입자 추적 소프트웨어를 DiaTrack (Semasopht 사용하여 측정 할 수 있습니다 http://www.microscopy.info/Organization/Details/4423; 박사 . 파스칼 Vallotton : http://www.csiro.au/en/Organisation-Structure/Divisions/Mathematics-Informatics-and-Statistics/PascalVallotton.aspx # A4). 이러한 속성은 S2 세포에게 조직 배양 세포의 미세 소관을 따라화물 수송을 연구 매력적인 시스템을 확인합니다. S2 세포에서의 파일럿 실험은 초파리 차 뉴런에서화물 운송 연구의 중요한 생리 학적 모델로 확장 될 수있다. S2 세포와 유사하게, 해리 된 배아로부터 배양 된 신경 세포는 염료와 같은 작은 분자 억제제에 대한 투과성 및 소기관 수송의 고해상도 실시간 이미징 실온에서 뉴런에서 수행 될 수있다. 다른 유전 적 배경을 가진 초파리를 사용하여, 우리는 녹아웃 (유전자 기능 상실 돌연변이)에 의해 기본 뉴런 유전자의 기능을 검사 할 수 있습니다, 최저 (dsRNA를 주입 또는 표현) 또는 자궁외 식 (유전자 변형 파리). 특히, CytoD 치료를 사용하여 액틴 필라멘트의 분열은 신경 돌기의 파생물을 방지하지 않는 대신 그들은 빠르게 성장하고, 따라서 우리는 미세 소관 depende를 공부할 수 있습니다굴지의 영향없이 CytoD 처리 된 신경 세포 NT 소기관 전송 11 필라멘트. 따라서, 기본의 연결을 문화는 생리 관련 시스템 (10, 12)에화물 운송을 연구 할 수있는 좋은 시스템을 만드는, 조직 배양 세포의 장점을 결합하고 유전자를 비행. 여러 가족 퇴행성 신경 질환에 의해 발생 또는화물 수송 결함 (예를 들면 파킨슨 병 (13)와 연관 될 수 있기 때문에, 헌팅턴 병 (14), 알츠하이머 병 (15, 16), 루게릭 병 / ALS 17 HMN7B 및 페리 증후군 18, 19, 및 척수 소뇌 실조증 유형 5/SCA5 20), 기본의 연결을 문화가 미세 소관 의존 전송에이 질병을 일으키는 특정 돌연변이의 효과 분석에 유용 할 수 있습니다. 마지막으로, 미세 소관에 의존하는 교통의 중요한 속성은 잘 포유 동물과 파리 사이에 보존되지만, <em&gt; 초파리 유전자 및 세포 배양 정상 및 병리학 적 상태에서 소기관 수송의 필수적인 측면을 연구하기위한 배양 된 초파리 세포의 사용을 정당화하는, 덜 비싸고 시간 소모적이다.

<table border="0" cellpadding="0" cellspacing="0" width="435">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Powder Insect cell medium (Schneider&#8217;s)</td>
			<td style="width:133px;">Sigma&#160;</td>
			<td style="width:179px;">S9895</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Insect &#8211;Xpress medium</td>
			<td style="width:133px;">Fisher</td>
			<td style="width:179px;">BW12730Q</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Insulin</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">I6634</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Penicillin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">P93000</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:124px;">Streptomycin</td>
			<td style="width:133px;">Research Products International</td>
			<td style="width:179px;">S62000</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Tetracycline hydrochloride</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">T7660-5G</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Fetal bovine serum</td>
			<td style="width:133px;">Atlanta Biologicals</td>
			<td style="width:179px;">S11150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Concanavalin A</td>
			<td style="width:133px;">Sigma</td>
			<td style="width:179px;">C2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:124px;">Cytochalasin D</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">IC15077105</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">LysoTracker Red DND-99</td>
			<td style="width:133px;">Molecular Probes</td>
			<td style="width:179px;">L7528</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">100 &#181;m Cell strainer</td>
			<td style="width:133px;">Fisher Scientific</td>
			<td style="width:179px;">22363549</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">25 mm Circle cover glass</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">48380 080</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:124px;">Drosophila Vials</td>
			<td style="width:133px;">VWR</td>
			<td style="width:179px;">89092-730</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:124px;">Disposable pellet pestles with matching microtubes</td>
			<td style="width:133px;">Kimble Chase</td>
			<td style="width:179px;">749520-0000</td>
		</tr>
	</tbody>
</table>

organelle transport in cultured drosophila cells: s2 cell line and primary neurons.

초파리 S2 세포는 균일하게 편광 된 미세 소관으로 가득 어떠한 액틴 해중합 약물 형태 긴 측쇄 프로세스의 존재 커버 슬립에서 도금했다. 세포 기관은 미세 소관 모터에 의해 이러한 프로세스를 따라 이동합니다. 쉬운 유지 보수, RNAi를 매개 단백질 노크 다운 및 안정적인 세포 라인을 만들기위한 효율적인 절차와 높은 감도는 초파리 S2 세포에게 라이브 영상으로화물 운송을 연구하는 이상적인 모델 시스템을 확인합니다. 해리 초파리 배아로부터 배양 된 신경 세포의 축삭 차 수송 : S2 세포로 얻어진 결과는 더욱 더 생리학 관련 시스템에 적용될 수있다. 배양 된 신경 세포는 S2 프로세스와 매우 유사 번들 미세 소관, 가득 긴 신경 돌기 성장. 같은 S2 세포에서 배양 된 신경 세포 소기관이 세포 내 소기관 중 하나를 특정 형광 염료 또는 DNA에 의해 암호화 형광 세포 기관 마커를 사용하여 시각화 할 수있는 초기 배아에 주입 또는 transgen 표현IC는 파리. 따라서 소기관 수송 간단 리빙 이미징을 사용하여 유리 커버 슬립에서 배양 된 신경 세포에서 기록 될 수있다. 여기에서 우리는 초파리 S2 세포 및 기본 뉴런에화물 운송을 배양하고 시각화하는 절차에 대해 설명합니다. 우리는 이러한 프로토콜은화물 수송을 연구 실험실에 대한 두 시스템에 액세스 할 수 있도록 믿습니다.

플랫 라운드 &quot;팬케이크&quot;모양 (그림 1A와 1C)에 CONA 코팅 coverslip에 확산에 부착 초파리 S2 세포. S2 세포 CytoD의 존재의 coverslips에 도금하고 2-3 시간 동안 확산을 허용하면 이들은 병렬 미세 소관 (도 1B 및 1D)로 채워진 얇고 긴 프로세스를 형성한다. 7을 플러스 끝이 미세 소관은 균일 한 극성이 있습니다. 초파리 S2 안정적 mCherry-튜 불린...

Watch this Scientific Journal Video about Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons. at JoVE.com

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

초파리 S2 세포 배양 신경 세포가 생체 내에서 모터 구동 세포 기관 수송의 이미징을위한 좋은 시스템입니다. 여기에서 우리는 두 종류의 세포를 배양에 대한 자신의 이미징 및 교통 분석을 상세한 프로토콜을 설명합니다.

교양있는 세포 기관 전송<em&gt; 초파리</em&gt; 셀 : S2 세포주 및 기본 뉴런.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

organelle-transport-cultured-drosophila-cells-s2-cell-line-primary

Research

JoVE Journal

Biology

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

13.6K 조회수.  Feinberg School of Medicine, Northwestern University.  초파리 S2 세포 배양 신경 세포가 생체 내에서 모터 구동 세포 기관 수송의 이미징을위한 좋은 시스템입니다. 여기에서 우리는 두 종류의 세포를 배양에 대한 자신의 이미징 및 교통 분석을 상세한 프로토콜을 설명합니다. 

비디오: Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

늦은 단계 Drosophila Pupae의 두뇌에서 기본의 연결을 문화

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

연구

생물학

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

기본 교양 Hippocampal 신경에 덴스 코어 Vesicles의 라이브 영상

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

의 준비 Drosophila</em라이트 현미경을 위해> S2 세포

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

교양 차 마우스 배아 섬유아 세포의 신호 분자의 아데노 바이러스 - 매개 유전자 제거

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

에서 기본 셀 문화 Drosophila Gastrula의 태아

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

쥐 간장 세포의 분리 및 차 문화

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

이미징 로컬 CA<sup&gt; 2 +</sup&gt; 배양 된 포유 동물 세포에서 신호

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a &quot;Humanized&quot; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

&quot;인간 답게&quot;를 생성<em&gt; 초파리</em키네신-5의 약리 억제에&gt; S2 세포주 민감성

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

포도당 섭취량 측정 및 인슐린 자극에 대한 반응<em&gt; In Vitro</em&gt; 배양 된 사람의 주요 근육 세포

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

기본 교양된 Murine Enterochromaffin 셀의 전체 셀 전기 생리학

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...