NZ, 동정 뉴런을 해부하고 수확에서 그녀를 훈련 연구소 회원 Subhas Biswas, 앤드류 스프 라울, 그리고 라이언 윌렛 감사하고 싶습니다. NIH - NINDS에서 교부금에 의해 지원됩니다.

1. 해부를위한 준비 : <ol><li> 적절한 문화 실험의 성격에 따라 접시 (10cm 또는 6 - 글쎄 또는 24 - 잘 접시 등) 및 외투들을 쥐 - 꼬리 콜라겐 24시간 절개 전에로를 선택합니다. 쥐 - 꼬리 콜라겐을 준비하고 외투 문화 요리에 사용하기위한 방법은 (1) 다른 설명했습니다. </li><li> 인산염의 0.25 % 트립신의 솔루션을 준비 호수 (PBS) (없음 EDTA (에틸렌 다이아 민 테트라 초산))와 필터 소독이 버퍼. -20 º C. 1 ML aliquots 및 상점 준비 </li><li> 소주 / deionized H 2 O. 2.5 MM 유리딘 각각 5 - FDU를 포함하는 솔루션을 준비 필터 - 소독 50 μl aliquots를 준비합니다. -20 º C.에 저장 </li><li> 10 %의 열 inactivated 말 혈청 (열 30 분 56 º C waterbath의 잠복기에 의해 inactivate)와 5% 태아 소 혈청과 RPMI 1640 배지 : 완전한 매체를 준비합니다. </li><li> 최종 중간 준비 : 1 %의 열 inactivated 말 혈청 + NGF (100ηg/ml 최종 농도) + 페니실린 / 스트렙토 마이신 (1 / 250) + 250분의 1 uridine/5-FDU 솔루션 RPMI 1640 배지 (10 μm의 최종 농도) . 참고 : 매번 사용하기 전에이 매체는 신선한까지하여야한다. 24 웰 플레이트 형식의 실험, 이러한 매체 12 ML 준비를해야합니다. 그것은 당신이 약간 여분을 준비하는 것이 좋습니다. NGF는 상업 다양한 소스 (이 프로토콜의 끝에 표 참조)에서 구입할 수 있습니다. </li><li> 장비 : 하나는 해부 현미경과 광원이 필요합니다. 초점 팁을 듀얼 구스넥 광섬유 조명기을 선호합니다. 스테인레스 포셉을 마이크로는 - 해부 두 켤레와 해부 가위 (또한 스테인레스 스틸 : 또한, 하나는 (autoclaving 또는 최소한 20 분 동안 70 %의 에탄올에 몸을 담글 수로) 다음과 같은 해부 도구를 청소하고 소독해야합니다 그리고 Roboz에서 모두). 두 무균 주사기 바늘은 (26 gague X 1 ½ 인치 또는 이와 유사한 것)도 필요합니다. 그것은 알루미늄 호일에 싸여 Kimwipe으로 덮여 스티로폼의 3 &quot;X3&quot;조각 해부 보드를 설정하는 것이 필요합니다. 오염을 제거하다 70 %의 에탄올과 보드를 스프레이. 마지막으로, 불타는 광택 유리 파스투레 피펫을 준비합니다. 회전하는 동안 화재 - 폴란드어하기 위해, 유리 피펫을 가지고 몇 초 동안 불꽃의 팁을시오. 검사시 팁은 매끄러운 모양과 개방이 좁아 것입니다. 이것은 세포가 dissociated 될 단계가 필요합니다. 피펫은 멸균 남아 있도록 세포 배양 후드에 불을 연마를 수행합니다. </li></ol> 2. 우수한 자궁암 신경의 해부 : <ol><li> 연령 P0 또는 P1의 아르 신생아 쥐의 새끼를 수집합니다. </li><li> 신속하게 오염의 가능성을 줄이기 위해 70 %의 에탄올과 해부를위한 강아지를 닦아냅니다. </li><li> 신속하게 가위 날카로운 쌍 (이 단계는 동영상에 표시되지 않습니다)와 새끼 목을 베다. 간단히 초과 혈액을 유출하기 위해 멸균 거즈에 대한 살균 집게를 사용하여 머리를 잡아. </li><li> 최대 직면하고있는 복부 측면과 당신을 향한 지향 꼬리 끝에있는 해부 패드에 머리를 놓으십시오. 절단 기관은 쉽게 볼 수 있습니다. 패드에 입천장하지만 한 핀을 밀어와 패드로 절단기도하지만 두 번째 핀을 눌러 다음 방법으로 해부 패드로 잘린 머리를 확보하기 위해 멸균 주사기의 바늘을 사용합니다. 해부 현미경으로 패드를 장소와 절개를 시작합니다. </li><li> 두 포셉 (각 손에 한)기도 주위 치워 피부와 지방을 사용하여 너무 깊이 가지 않도록주의한다. 이렇게하면 기관의 양쪽에 서로 거의 평행 실행 근육의 한 켤레를 노출합니다. 참고 : 해부하는 동안, 당신이 추위 멸균 PBS 또는 추위, 과도한 혈액을 멀리 세척하고 지역 깨끗하고 촉촉한 유지 살균, 파스퇴르 피펫과 함께 제공되는 혈청 무료 RPMI 1640 배지와 관개해야합니다. 이것은에 따라 단 한 번 이상 할 수 있습니다 얼마나 빠른 걸로 작동합니다. 경험 해부학자 들어, 신경 한 켤레를 해부하다에만 대해 두 3 분 걸립니다. </li><li> 첫 번째 측면에있는 근육을 절단하고 제거하는 포셉을 사용합니다. </li><li> 근육이 제거되면, 경동맥이 표시됩니다. 종종 그것은 밝은 분홍빛이 도는하고 혈액을 포함하고 있습니다. 그것은 당신이 주변에서 볼 수있는 다른 혈관보다 색상의 가벼운 나타날 수 있으며 이런 이유 경동맥 바로 눈에 띄지 않을 수도 있습니다. 이 동맥은 &quot;Y&quot;모양의 리본 생겼는지으로기도와 bifurcates 함께 실행됩니다. 이 분기점은 중요한 이정표이며이있는 한, 그것은 SCG를 찾을 상대적으로 쉽게됩니다. </li><li> 가장 꼬리 끝에있는 동맥을 끊어와 포셉 (상대방이 여전히 연결된)를 약간 들어올려. 일단 이렇게, 당신은 thr 느슨하게 단지 분기점의 시점에서 동맥에 붙어 있으며 조직의 아몬드 모양, 광택 찾고 작은 질량을 가지고 볼 수 있습니다사전 및 / 또는 사후 ganglionic connectives EAD 같은. 이것은 우수한 경추 신경절이다. 부드럽게 감기, 혈청 무료 RPMI 1640 매체를 포함하는 작은 문화 요리 (35 ㎜)에과 장소를 분리하는 반면에 포셉의 두 번째 쌍을 사용합니다. </li><li> 다음, 같은 프로토콜을 다음과 같은 기관의 다른 측면에 신경을 압축을 풉니다. </li></ol> 3. 신경 청소 : <ol><li> 일단 모든 신경이 밖으로 해부되었습니다, 그들은 최대한 관계없는 조직을 해제해야합니다. </li><li> 해부 현미경 아래에서, 당신은 신경이 경동맥, FAT 또는 기타 조직의 조각을 첨부 것을 볼 수 있습니다. 이러한 불필요한 물질을 멀리 애타게 각 손에 집게와 함께 작동합니다. 장소는 RPMI 1640 매체를 포함, 살균 15ml 원뿔, 폴리 프로필렌 튜브에 신경을 청소. </li><li> 사전 및 / 또는 사후 ganglionic connectives도 멀리 줄어들 수 있습니다. 각 신경절은 약 1 만 뉴런 있습니다. 뉴런은 도금되면, 그들은 neurites를 regrow 것입니다. </li></ol> 4. 교감 신경의 문화를 구축 : <ol><li> 이분에 대한 200xg에 신경을 원심과 뜨는 폐기하십시오. </li><li> 0.25 %의 트립신 솔루션의 0.5-1 ML에 신경을 Resuspend. 30 분 37 ˚ C waterbath이나 보육에 놓습니다. 이것은 신경의 세포를 떼어 놓다 도움이 될 것입니다. </li><li> 30 분 후에 밖으로 희석 2 분 200xg에서 트립신과 원심 분리기를 무력화하기 위해 완전한 중간의 10 ML를 추가합니다. </li><li> 뜨는 최종 중간 2 ML에 resuspend 폐기하십시오. </li><li> 더욱 불을 광택 유리 파스투레 피펫으로 신경을 위아래 triturating하여 세포를 떼어 놓다. 신경 20 번 씹다하고 몇 분 동안 얼음에 튜브를 놓으십시오. </li><li> 한편, (약 3분의 2 1 / 2mm 지름) 자리가 좁아하기 위해 더 많은 화재 - 폴란드어 피펫. 그것이 냉각까지 몇 분 기다려 20 배 더 신경을 씹다. 계속으로, 매체는 점차적으로 cloudier 세포 dissociated되고있다는 것을 나타내는 될 것입니다. 한 두 번 더 세포 분열시키다 얼마나 잘에 따라이 단계를 반복합니다. 시간 후, 당신은 모든 신경은 뉴런에 dissociated하고 더 완전한 신경은 튜브에서 볼 수없는 것을 통지합니다. 교제를 끊다되지 않습니다 일부 조직 물질이있을 수 있습니다. 이 시점에서 어떤 undissociated 파편이 바닥에 해결 할 수 있도록 튜브가 몇 분 동안 얼음에 앉아 보자. 이제 조심스럽게 다른 튜브에서 최고의 장소에서 거의 모든 매체를 수집합니다. 당신이 undissociated 파편을 수집하지 않도록하기 위해 튜브의 하단에있는 약 50 μl를 떠나. dissociated 세포를 포함하는 튜브에 최종 매체를 더 추가 :이 볼륨 문화 플레이트 하나가 문화에 신경을 사용하는의 종류에 따라 다릅니다. 예를 들면 다음과 같습니다 잘 따라 최종 매체의 0.5 ML에 잘 당 24 잘 요리가 직업이있다면, 그때 플레이트 0.5 신경 (물론 당 ~ 10,000 뉴런). 전형적인 쥐 쓰레기는 24 신경 줄 래요 12 새끼,로 구성되어 있습니다. 이 종자 일 24 잘 플레이트 및 최종 중간의 총 부피 12 ML입니다 충분하다. 참고 : 신경이 문화에 제공됩니다 glia와 다른 세포 유형의 작은 인구에도 포함되어 있습니다. 최종 매체 Uridine/5-FDU를 사용하면 자신의 확산을 방지하고 그들의 죽음을 촉진합니다. </li><li> 피드 세포 신선한 RPMI 1퍼센트 말 혈청 + NGF와 uridine/5-FDU과 함께 매체의 2 / 3를 대체하여 모든 48시간. 참고 : 처음에는 문화가없이 neurites와 둥근 모양 교감 신경이 포함되어 있습니다. 짧은 neurites는 절개 후 보이는 24 시간이되고 점차적으로 밀도가 더 시간이 지남에 따라 분기가됩니다. </li></ol>

<ol>
	<li>Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , (1998).</li><li>Biswas, S. C., Shi, Y., Sproul, A., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-apoptotic+Bim+Induction+in+Response+to+Nerve+Growth+Factor+Deprivation+Requires+Simultaneous+Activation+of+Three+Different+Death+Signaling+Pathways.">Pro-apoptotic Bim Induction in Response to Nerve Growth Factor Deprivation Requires Simultaneous Activation of Three Different Death Signaling Pathways.</a> The Journal of Biological Chemistry. 282 (40), 29368-29374 (2007).</li><li>Bocchini, V., Angeletti, P. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nerve+Growth+Factor:+purification+as+a+30,000-molecular-weight+protein.">The Nerve Growth Factor: purification as a 30,000-molecular-weight protein.</a> Proc. Natl. Acad. Sci. USA. (64), 787-794 (1969).</li><li>Dechant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chat+in+the+Trophic+Web:+NGF+Activates+Ret+by+Inter-RTK+Signaling.">Chat in the Trophic Web: NGF Activates Ret by Inter-RTK Signaling.</a> Neuron. 33 (2), 156-158 (2002).</li><li>Deckwerth, T., Elliott, J. L., Knudson, C. M., Johnson, E. M., Snider, W. D., Korsmeyer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bax+is+required+for+neuronal+death+after+trophic+factor+deprivation+and+during+development.">Bax is required for neuronal death after trophic factor deprivation and during development.</a> Neuron. (17), 401-411 (1996).</li><li>Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+in+Vitro+Studies+on+the+Nerve+Growth+Factor+(NGF)+Requirement+of+Neurons+-+I.+Sympathetic+Neurons.">Quantitative in Vitro Studies on the Nerve Growth Factor (NGF) Requirement of Neurons - I. Sympathetic Neurons.</a> Developmental Biology. (58), 96-105 (1977).</li><li>Park, D. S., Morris, E. J., Padmanabhan, J., Shelanski, M. L., Geller, H. M., Greene, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin-dependent+kinases+participate+in+death+of+neurons+evoked+by+DNA-damaging+agents.">Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.</a> The Journal of Cell Biology. 143, 457-467 (1998).</li><li>Pierchala, B. A., Ahrens, R. C., Paden, A. J., Johnson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+Growth+Factor+Promotes+the+Survival+of+Sympathetic+Neurons+through+the+Cooperative+Function+of+the+Protein+Kinase+C+and+Phosphatidylinositol+3-Kinase+Pathways.">Nerve Growth Factor Promotes the Survival of Sympathetic Neurons through the Cooperative Function of the Protein Kinase C and Phosphatidylinositol 3-Kinase Pathways.</a> The Journal of Cell Biology. 279, 27986-27993 (2004).</li></ol>

The authors have nothing to disclose.

우리는 우리의 문화 스프 라그 돌리 쥐를 사용합니다. 신경 청소 때 시간과주의를 가져가라. 모든 파편, 혈관과 지방을 제거합니다. 이 단계는 더 많거나 적은 균일한과 관계없는 세포 종류의 무료 문화 확보에 중요합니다. uridine/5-FDU의 추가는 크게 문화를 오염 나머지 (mitotic) 이외의 연결을 세포를 제거하거나 적어도 증식을 억제합니다. 또한, 분리 단계 동안 인내심을하고는 접시에 씨앗되면 그들?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Forceps, Stainless steel #5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS4976</td>
<td>&nbsp;</td>
<tr>
<td>Scissors, DEAVER straight sharp-blunt- 43mm blades - 5.5</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS6760</td>
<td>&nbsp;</td>
<tr>
<td>RPMI 1640 w/ L-Glutamin</td>
<td>Reagent</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>10-040-CV</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>Reagent</td>
<td>SAFC Biosciences</td>
<td>12103c</td>
<td>&nbsp;</td>
<tr>
<td>Donor Horse Serum</td>
<td>Reagent</td>
<td>JRH Biosciences</td>
<td>12449</td>
<td>Heat-inactivate by incubating in 56&deg;C waterbath for 30 minutes.</td>
</tr>
<tr>
<td>Penicillin/streptomycin</td>
<td>Reagent</td>
<td>Gibco, Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin without EDTA</td>
<td>Reagent</td>
<td>Difco</td>
<td>MT 25-050-CI</td>
<td>&nbsp;</td>
<tr>
<td>Uridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>U3003</td>
<td>&nbsp;</td>
<tr>
<td>5-Fluoro-5' deoxyuridine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F-8791</td>
<td>&nbsp;</td>
<tr>
<td>NGF</td>
<td>Reagent</td>
<td>Harlan Bioproduct</td>
<td>BT-5025</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Microscope and light source</td>
<td>Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 ml polypropylene tubes</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>352096</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes</td>
<td>Tool</td>
<td>Corning, Nunclone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

protocol for culturing sympathetic neurons from rat superior cervical ganglia (scg)

쥐에서 우수 자궁 경부 신경 (SCG)은 교감 신경을 포함 작은 광택, 아몬드 모양의 구조입니다. 이들 뉴런은 머리와 목 지역에 대한 공감 innervations를 제공하고 그들은 잘 특징과 비교적 균질 인구 (4) 구성합니다. 교감 신경이 NGF의 가용성은 그들의 문화와 실험 조작 (2, 3, 6)을 용이하게 생존, 분화 및 axonal 성장과 넓은 확산에 대한 신경 성장 인자 (NGF)에 따라 달라집니다. 이러한 이유로, 교양 교감 신경이의 연결을 개발 및 분화, 프로그램 및 병리 학적 세포 죽음의 메커니즘 및 신호 전달 (1, 2, 5, 6)를 포함하여 연구의 다양한 사용되었습니다. 신생아 쥐 및 culturing 교감 신경에서 SCG를 해부하는 것은 매우 복잡하지 않으며 매우 빠르게 마스터하실 수 있습니다. 이 문서에서는, 우리는 신생아 쥐 새끼에서 SCG을 해부하다하고 교감 신경의 문화를 확립하기 위해 그들을 사용하는 방법을 자세히 설명합니다. 이 문서는 또한 준비 단계와이를하는 데 필요한 다양한 시약과 장비를 설명합니다.

Watch this Scientific Journal Video about Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG at JoVE.com

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

이것은 갓 태어난 새끼 쥐의 우수한 경추 신경 (SCG)에서 기본 교감 신경을 분리하고 문화하는 방법을 설명하는 프로토콜입니다.

쥐 우수한 자궁암 신경 (SCG)에서 Culturing 교감 신경에 대한 프로토콜

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

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Biology

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

22.6K 조회수.  Columbia University. 이것은 갓 태어난 새끼 쥐의 우수한 경추 신경 (SCG)에서 기본 교감 신경을 분리하고 문화하는 방법을 설명하는 프로토콜입니다.

비디오: Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

Microfluidic 장치에서 Compartmentalization에 대한 E18 두피 랫 뉴런 준비

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

연구

생물학

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

호중구 격리 프로토콜

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

Compartmented 문화의 도설 루트 신경의 뉴런의 준비 및 유지 관리

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

웜 실험실 소개 : Culturing 웜에서 돌연변이 유발하는

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

야생 유충에서 Hemocytes을 추출하기위한 간단한 프로토콜

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Culturing Primary Rat Inner Medullary Collecting Duct Cells

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP binds to vasopressin V2 receptors (V2R) on the surface of the cells and thereby induces synthesis of cAMP. This stimulates cellular signaling processes leading to changes in the phosphorylation of the water channel aquaporin-2 (AQP2). Protein kinase A phoshorylates AQP2 and thereby triggers the translocation of AQP2 from intracellular vesicles into the plasma membrane facilitating water reabsorption from primary urine. Aberrations of AVP release from the pituitary or AVP-activated signaling in principal cells can cause central or nephrogenic diabetes insipidus, respectively; an elevated blood plasma AVP level is associated with cardiovascular diseases such as chronic heart failure and the syndrome of inappropriate antidiuretic hormone secretion.
Here, we present a protocol for cultivation of primary rat inner medullary collecting duct (IMCD) cells, which express V2R and AQP2 endogenously. The cells are suitable for elucidating molecular mechanisms underlying the control of AQP2 and thus to discover novel drug targets for the treatment of diseases associated with dysregulation of AVP-mediated water reabsorption. IMCD cells are obtained from rat renal inner medullae and are used for experiments six to eight days after seeding. IMCD cells can be cultured in regular cell culture dishes, flasks and micro-titer plates of different formats, the procedure only requires a few hours, and is appropriate for standard cell culture laboratories.

Arginine-vasopressin (AVP) controls fine-tuning of body water homeostasis through facilitating water reabsorption by renal principal cells. Here, we present a protocol for the cultivation of primary rat inner medullary collecting duct cells suitable for the elucidation of molecular mechanisms underlying AVP-mediated water reabsorption.

덕트 세포를 수집 배양 차 쥐 내부 수질

Arginine-vasopressin (AVP) facilitates water reabsorption by renal collecting duct principal cells and thereby fine-tunes body water homeostasis. AVP ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

쥐의 측면 꼬리 정맥에서 혈액을 샘플링

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146의 분리<sup&gt; +</sup쥐 폐에서&gt; 주민 폐 중간 엽 기질 세포

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

마우스에서 췌장 췌도 격리를 위한 간단한 고효율 프로토콜

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

기계적 평가를 위한 인간 근육 티비아리스 전방의 우수한 구획에서 골격 근육 생검 의 컬렉션

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...