기금은 &quot;PROGETTO concorso 폰다 치오 IRCCS 오스 페달 마조 레 Policlinico 2009년부터 2010년까지&quot;MD, 그리고 Associazione 밤비노 Nefropatico ABN Onlus, 밀라노에 의해 제공되었다

모든 동물 실험은 국립 과학적인 목적을 위해 사용하고 검토와 밀라노 대학의 윤리위원회에 의해 승인 된 동물의 보호에 관한 유럽의 현재의 규정에 따라 수행되었다. 차 조골 세포의 1. 격리 상기 방법은, 사소한 수정으로, Dodig 등 (14)에 의해 기술 된 절차를 따른다. <ol><li> 0.1 % 콜라게나 제 P와 0.05 %를 추가하여, 매체를 소화 준비 HBSS 매체 (행크스 균형 소금 솔루션, 아니 칼슘, 마그네슘, 또는 페놀 레드)에 (2.5 % 트립신 10 배, 아니 페놀 레드에서 시작) 트립신. </li><li> 3~4일 이전 마우스를 사용하여 (절차 1 마우스 적은에 적용될 수있다.) </li><li> 잘린 희생. 70 % 에탄올로 머리를 살포하고 얼음에 보관. 핀셋과 해부 / 외과 가위로 피부와 근육 층을 제거합니다. </li><li> 부드럽게 정수리 뼈를 제거하고두 반쪽의 시상 봉합에 따라 구분합니다. 반드시 뼈를 봉합하고 자기편 조직 무료입니다 확인하고 절차가 완료 될 때까지 HBSS 매체를 포함하는 잘 접시에 개별 뼈 조각을 배치합니다. </li><li> HBSS를 취소하고 각 웰에 추가 37 ℃에서 15 분 동안 소화 매체 </li><li> 상층 액을 버린다. </li><li> 다시 37 ℃에서 15 분 동안 소화 매체를 추가 </li><li> 이 시간은 상등액을 저장하고 10 % FBS (태아 혈청)과 1 % 스트렙토 마이신 / 페니실린과 보충 α-MEM (최소 필수 중간)에 배치합니다. </li><li> 반복은 1.7 및 1.8 배 단계를 반복합니다. </li><li> 5 분 425g에 원심 분리하여 세 수집 분수, 스핀 세포 해변, 상층 액을 버린다 세포 펠렛을 재현 탁하는 보충 알파-MEM 3 ㎖를 추가하고, 전송 25cm 2 플라스크. </li><li> 파편과 죽은 세포를 제거하는 24 시간 후에 매체를 변경합니다. </li><li> 50 ㎍ / ㎖의 아스 코르 빈산 (AA)과 글리 3 밀리미터 추가cerol 2 - 인산이 나트륨 염 α-MEM 배지로 하이드레이트 (GP). </li><li> 그 후, 매일 (α-MEM 플러스 AA / GP) 매체를 변경합니다. </li></ol> 2. ATRA 프로토콜 <ol><li> 불 활성화를 방지하기 위해 어두운 방에 ATRA (모든 - 트랜스 레티노 산) 모든 실험을 실시한다. </li><li> HBSS 매체 1X 트립신 용액을 제조하고 사용할 때까지 37 ° C로 유지한다. </li><li> subconfluent 세포에서 매체를 제거하고 조심스럽게 HBSS로 세척. </li><li> 1 ML 트립신을 추가, 부드럽게 모든 세포는 트립신에 의해 보호 될 수 있도록 플라스크를 회전하고, 37 ℃에서 3 분 동안 품어 </li><li> 거꾸로 현미경으로 세포의 실제 분리를 확인합니다. </li><li> 트립신을 불 활성화하는 α-MEM 배지 3 ㎖를 추가합니다. </li><li> 세포를 복구하고 425g에서 15 분 동안 원심 분리. </li><li> 신선한 배지에서 세포 펠렛을 재현 탁. </li><li> 혈구 계산 챔버의 하락을 놓고, 몇 분 동안 정착 할 수 있도록하고 CE를 계산LL 번호. </li><li> 10,000 세포 / α-MEM 플러스 AA / GP와 cm 2의 밀도로 25cm 2 플라스크에 넣고 세포. 세포 밀도 세포 프로세스와 세포 간 접촉의 최적의 개발에 관련된주의하십시오. 초기 밀도 이상 10,000 세포 / cm 2가 세포 프로세스의 적절한 발달을 저해하지만, 낮은 세포 수로 인해 세포 간 접촉의 부재로, 조기 ​​세포 사멸의 원인. </li><li> 매 24 시간 매체에 5 μm의 ATRA를 추가합니다. </li><li> 형태 학적 변화는 일반적으로 48 시간 후에 관찰 : 형태를 평가하기 위해 매일 세포를 모니터링합니다. 제 24-48 시간 후에, 세포 증식이 정지되어있는 경우, 상기 밀도 replate. 성숙한 골 세포의 균일 한 인구 4 ~ 5 일 후에 존재합니다. 필요에 따라 12 ~ 15 일까지의 임의의 시점에서 전지를 사용한다. </li><li> 세포 (예 : 커버 슬립이나 잘 플레이트와 같은) 다른 지원에 도금하는 경우, 위의 밀도가 유지되어 있는지 확인하고사용할 때까지 ATRA마다 24 시간을 추가로 진행합니다. 실험하기 전에 적어도 24 시간에 대한 새로운 지원을 준수 세포를 둡니다. </li></ol> 3. 면역 염색 <ol><li> 면역 염색의 경우, 커버 슬립에 세포를 플레이트 (버리 (15) 예를 들어 참조) 설치 방법을 따르십시오. </li><li> 예를 들어, 간접 면역 형광에 대해, 30 분 동안 RT에서 어두운 이차 형광 표지 된 항체와 그 후, 가습 챔버 내에서 1 시간 동안 RT에서 일차 항체로 순차 부화, 4 % 파라 포름 알데히드 또는 차가운 아세톤으로 고정 가습 실에서. </li><li> 핵 counterstaining에 DAPI 또는 유사체를 사용하고 중간 안티 페이딩에 탑재합니다. </li></ol> 4. 알리자린 레드 염색 및 추출 <ol><li> 커버 슬립에 세포를 접시. </li><li> 10 분 동안 70 % 에탄올에 coverslip에 침지하여 세포를 고정합니다. </li><li> 알리자린 레드 염색 액의 일부 방울 (1 ㎎ / ㎖, pH를 5.5)와 함께 세포를 커버30 분. </li><li> 수돗물로 커버 슬립을 씻으십시오. 유리 슬라이드에 글리세롤의 드롭으로 장착하고 수직 배의 현미경 20X 배율을 사용하여 이미지를 가지고. </li><li> 위의 과정 후, 수돗물에 슬라이드를 담가 커버 슬립을 복구 할 수 있습니다. </li><li> 웰 플레이트의 각 coverslip에 넣습니다. </li><li> 60 % 과염소산 (양 완전히 세포를 커버 할 수있을 정도로 충분히 커야합니다)를 추가하고 교반과 함께 4 ° C에서 O / N 두십시오. </li><li> 450 nm 파장에서 분광 광도법에 의해 상층의 광학 밀도를 참조하십시오. </li><li> 세포 수에 대한 값을 정규화하기 위해, 10 분 동안 세포 제제 다루 야누스 그린 (PBS에 0.​​2 % 용액)를 추가한다. </li><li> 초순수로 조심스럽게 세척 할 것. </li><li> 0.5 N 염산을 (양 모든 세포를 커버 할 수있을 정도로 충분히 커야합니다)를 추가하고 몇 초 동안 손으로 가볍게 흔들어. </li><li> 분광 광도계로 615 nm에서 흡광도를 참조하십시오. </li></ol>

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저자가 공개하는 게 없다.

지난 몇 년 동안 골 세포가 뼈에서 가장 중앙 세포로 떠오르고있다. 연구의 발전은 점진적으로 뼈 질환의 다양한 소설과 더 나은 치료를 디자인하는 엄청난 가치가 이전에 의심받지 검증되지 않은 골 세포의 속성의 수를 공개하고 있습니다. 그러나, 골 세포 생물학의 연구는 골 세포와 같은 세포 라인 MLO-Y4가 제공되기 전에 조골 세포가 오랜 기간 동안 대리 세포로 사용되어왔다있을 정도로 체...

골 세포, 가장 풍부한 뼈 세포 유형은 깊게 골격 내에있는 고도로 분지의 말단 분화 세포이다. 성숙한 골 세포의 세포체는 뼈 lacunae에 포함되는 다양한 형태를 가지고있다; 둥근 골 세포가 뼈 소주 1에서 더 자주 반면 신장 세포 기관과 골 세포는 대뇌 피질의 뼈에서 발견된다. 분지 수상 돌기는 다른 골 세포와, 또한 다른 뼈 세포 유형, 골수, 혈관과 관련된 혈관 주위 세포와뿐만 아니라 여러 연락처를 만드는 복잡한 웹을 형성, 눈물 소관이라는 작은 채널에서 세포체에서 연장 상주합니다. lacunae 및 소관에 함유 간질 유체를 통해, 골 세포는 그러므로 지역뿐만 아니라 전신적인 이벤트 및 그 반대의 동작은 로컬 및 전신의 변화 (2)에 의해 조절 될 수뿐만 아니라 영향을 미칠 수있다, 또한 궁극적으로 순환 시스템에 연결된다. 골 세포의 연구는 최근, 이러한 조직과 세포 고유의 형질 전환 동물의 발생, 강력한 현미경 기술과 높은 처리량 분자 스크리닝 3,4의 사용과 같은 몇 가지 기술적 진보, 덕분에 힘을 얻고있다. 그러나, 이들 세포의 기술은 주로 시험 관내 모델에서의 적절한 상대적 부족에 여전히 불완전하다. 사실, 골 세포로 인해 깊은 위치의 획득과 문화에 유지하는 것이 곤란하고, 이러한 분화 된 세포 유형을 특징 짓는 확산의 낮은 수준했습니다. 그들은 일반적으로 낮은 세포 수율을 생산하고 심지어 몇 오염 아세포 급속 골 세포 8 자라다 할 위험을 가지고 있지만 년 함께, 다수의 방법은, 일차 5-7 골 세포를 분리하기 위해 개발되었다. 이러한 이유로, 체외 작업에 대부분의 실험은 잘 확립 된 골 세포의 세포 L에 지금까지 실시 된오프라인 MLO-Y4 9. 추가 시험 관내 방법이 세포 연구의 가능성을 향상시킬 수와 골 세포 생물학 및 병태 생리의 분석을 향상시킬 수 있습니다. 주로 채택 될 것으로, 이러한 방법은 쉽게 재현해야하며, 세포 성숙에 도달하기 위해 특수 장비 또는 매우 긴 시간이 필요하지 않을 것입니다. 일차 전지에 적용 할 경우 중요한 것은, 그들은 그것이 가능한 형질 전환 동물을 활용하도록 만들 것입니다. 우리는 최근 조골 세포주 MC3T3-E1 지점 레티노 산과 차 조골 세포의 치료는 골 세포 (10)의 형태 및 분자의 특징을 담 분지 셀 균질 집단의 발전을 선도 급속한 표현형의 변화를 유도하는 것을 설명했다. 모든 - 트랜스 레티노 산 (ATRA)는 핵 레티노 산 수용체 (RAR들)을 결합하여 유전자 전사를 조절하는 비타민 A의 활성 대사 제품입니다. RAR의궁극적으로 레티노 산 (RA) 응답 표적 유전자 (11)의 변조로 이어지는, 레티노이드 X 수용체 (RXRS)와 이종로 DNA에 결합. ATRA는 신경 세포 (12)와 족 세포 (13)와 같은 다른 분지의 세포가 그 (것)들의 사이에서, 여러 종류의 세포 분화와 성숙을 조절하는 것으로 알려져있다. 여기서 설명한 방법은 일차 골아 세포에 ATRA의 첨가에 기초한다. 효과적이기 위하여, ATRA는 정의 된 밀도에서 도금 셀에 정확한 성숙 단계에서 첨가 할 수있다; 우리의 경험에서 이러한 조건은 조골 세포의 골 세포로 표현형의 스위치를 얻을 중요합니다.

<table border="0" cellpadding="0" cellspacing="0" style="width:923px;" width="923">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:283px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:219px;">Cat #</td>
			<td style="width:183px;">comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase P</td>
			<td>Roche Applied Science</td>
			<td>11213857001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Gibco, Life Technologies</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Gibco, Life Technologies</td>
			<td>14175129</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alpha-MEM</td>
			<td>Invitrogen, Life Technologies</td>
			<td>22571038</td>
			<td>Pre-warm at 37&#176;C before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin/Penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol 2-phosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ATRA</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
			<td>Protect ATRA from light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Dissolve in PBS and filter before use. Work always under a chemical hood.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>32670</td>
			<td>Can be added to the secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alizarin red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Janus Green</td>
			<td>Sigma-Aldrich</td>
			<td>201677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>176745</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Use with caution (skin and eye protection are recommended)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">fluorsave</td>
			<td>Calbiochem - Merck</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #7, 0.40 x 0.5mm tips</td>
			<td>World Precision Instruments</td>
			<td>501981</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Economy Tweezers #4, 0.40 x 0.45mm tips</td>
			<td>World Precision Instruments</td>
			<td>501978</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors, straight,10cm curved</td>
			<td>World Precision Instruments</td>
			<td>14394</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Scissors, 14 cm, straight, S/S</td>
			<td>World Precision Instruments</td>
			<td>501218</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Culture flasks</td>
			<td>Corning</td>
			<td>430168</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Well-plates</td>
			<td>Corning</td>
			<td>3335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermanox coverslips</td>
			<td>Thermo Scientific Nunc</td>
			<td>12-565-27</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">microscope</td>
			<td>Zeiss Apotome</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">spectrometer</td>
			<td>Safas Xenius</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

application of retinoic acid to obtain osteocytes cultures from primary mouse osteoblasts

<html> 골 세포 배양의 필요성은 잘 뼈 연구원의 지역 사회에 알려져있다; 기본 골 세포의 분리가 어렵고, 낮은 세포 수를 생산하고 있습니다. 따라서, 가장 광범위하게 사용되는 셀룰러 시스템은 골 세포 조의 MLO-Y4 세포주이다. 여기에서 설명하는 방법은 형태 학적 및 분자 골 세포의 기능과 분지 세포의 균일 한 인구를 생성하는 레티노 산의 사용을 말합니다. 쥐의 두개골로부터 골아 세포의 분리 후, 모든 - 트랜스 레티노 산 (ATRA)이 세포 배지에 첨가하고, 셀 모니터링 도립 현미경으로 매일 수행된다. 첫 번째 형태 학적 치료와 차별화 2 일 후에 탐지 가능하면 수상 돌기 세포 외 기질을 생산하는 능력의 손실, 조골 세포 마커의 다운 규제의 점진적 발전과 함께 5 일에서 일반적으로 완료까지 규제 골 세포 특정 분자의. 콘텐츠는 &quot;&gt; 일일 셀 모니터링 때문에 일차 세포의 고유 변동의 필요, 및 프로토콜은 상이한 마우스 균주로부터 얻어진 형질 전환 모델에 적용된 셀에 최소 변화에 적응 될 수있다. 방법은 수행하기 쉽고, 특수 장비를 필요로하지 않으며, 이는 높은 재현성으로, 급속 무제한 생물학적 응용을위한 이들 세포의 사용을 허용하는, 세포 외 기질의 완전한 부재 하에서 성숙한 골 세포 집단을 생성한다.

결과는 5 개의 독립적 인 실험 10을 얻었다. 세포 형태 AA / GP는 일차 전지는 대부분 조약돌 같은 기능, 성숙한 조골 세포의 특성을 가지고 처리. 산재 분지 세포 (그림 1A에서 빨간색 화살표로 표시된)을 가능성이 골 세포를 대표하는 찾을 수 있습니다. ATRA 치료로, 세포는 빠르게 일반적으로 이일 후 관찰되는 파급 효과를 표?...

Watch this Scientific Journal Video about Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts at JoVE.com

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

레티노 산으로 기본 마우스 조골 세포의 치료는 골 세포의 형태 학적 및 분자 기능 베어링 분지 세포의 균일 한 인구를 생산하고 있습니다. 방법은 획득 및 배양 주 골 세포를 유지의 어려움을 극복하고, 형질 전환 모델 유래 세포를 연구하는 것이 유리할 수있다.

기본 마우스 조골 세포에서 골 세포 배양을 구하는 레티노 산의 적용

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

application-retinoic-acid-to-obtain-osteocytes-cultures-from-primary

Research

JoVE Journal

Biology

13.3K 조회수.  Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico.  레티노 산으로 기본 마우스 조골 세포의 치료는 골 세포의 형태 학적 및 분자 기능 베어링 분지 세포의 균일 한 인구를 생산하고 있습니다. 방법은 획득 및 배양 주 골 세포를 유지의 어려움을 극복하고, 형질 전환 모델 유래 세포를 연구하는 것이 유리할 수있다. 

비디오: Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

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

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

쥐 Neonates에서 기본 Dissociated Midbrain의 도파민 세포 문화

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Electroporation에 대한 Murine 골수에서 조혈 세포 차 문화의 준비

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

설치류에서 기본 성인 Fibroblast 문화를 구축

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

유도 만능의 줄기 세포에서 파생 마우스의 생성

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

마우스의 심장에서 차 멜라닌 세포와 같은 세포를 분리

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

마우스와 인간의 대장 조직에서 주요 근육 섬유 모세포의 분리

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.&#160;teleost fish) and full regeneration following appendage loss (i.e.&#160;urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4.

In vitro culture systems have proven indispensible to our understanding of vertebrate myogenesis. However, much remains to be learned about nonmammalian skeletal muscle development and growth, particularly in basal taxa. An efficient and robust protocol for isolating the adult stem cells of this tissue, the myogenic precursor cells (MPCs), and maintaining their self-renewal, proliferation, and differentiation in a primary culture setting allows for the identification of conserved and divergent regulatory mechanisms throughout the vertebrate lineages.

기초 척추 계통의 주요 근원 성 전구 세포 / 근원 세포 배양의 준비

Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem ...