1。细胞分离<ol><li>流程图，概述了协议，显示图1。特别是在本协议所使用的试剂的目录编号，请参考表1。剖析在PBS洗澡的视网膜。在解剖中，最好是删除的，因为玻璃体视网膜可以阻碍分离和镜头。它并不总是非常重要的，以消除所有的视网膜色素上皮（RPE），并在某些情况下，它可能无法彻底清除它。然而，感光细胞的单细胞分析实验中，RPE应该被删除。未能取出视网膜色素上皮可导致污染的视杆细胞与视网膜色素上皮基因表达概况。这大概是由于感光细胞和视网膜色素上皮之间的连接。 分离，整个成年小鼠视网膜孵育10分钟，含20在1.5 ml离心管在37°C辅以10毫米肝素钠激活木瓜蛋白酶μL，20μL25毫米5毫米EDTA半胱氨酸，360μL汉克的平衡盐溶液（HBSS中）。用于过滤嘴的枪头整个协议的所有步骤，以尽量减少潜在的污染。木瓜蛋白酶浓度和孵育时间将根据不同的年龄和组织的性质。例如，以游离在产后当天（P0）分离视网膜，孵化10μL激活木瓜蛋白酶，10μL25 5 mM的EDTA的mM的半胱氨酸，与380μL汉克的平衡盐溶液（HBSS中）辅以10毫米肝素钠视网膜在37°C为10分钟来自不同物种的视网膜也需要略有不同的条件。鸡视网膜是比大多数的发展阶段小鼠视网膜更薄。使用10μL激活木瓜蛋白酶，10微升25毫米5毫米EDTA半胱氨酸，380μL汉克的平衡盐溶液（HBSS中）以10毫米为5分钟肝素钠，在37°C间足够的游离ţ这些视网膜。 </li><li>轻轻拍打管驱逐任何解决的细胞，然后磨碎轻轻的10-20倍，与P1000移液器。额外的10分钟孵育在37°C，如果坚持大的组织团块，然后再重新磨碎的10-20倍。 </li><li>加入5μLDNA酶I（10 U /μL），静置室温5分钟。与P1000移液器轻轻磨碎。时代的确切数目将需要凭经验确定，但一般落在10-20之间。 </li><li>在表上的离心机离心3分钟3000转，取出上清液，留在底部100-200微升的液体，挖掘管打跑沉淀。加入1毫升的HBSS和悬浮颗粒，温和研磨。攻管是关键，因为这里只是重新悬浮颗粒与许多视网膜细胞裂解。这是用成年鼠的视网膜尤其如此。 </li><li>在3分钟3000转离心，小心取出450μL的PBS上清，重悬包含0.1％的牛血清白蛋白。重要的是尽可能上清删除，以尽量减少从裂解细胞，并从任何产品的分离，可以抑制未来的反应的基因存在。 </li></ol> 2。收获的单细胞<ol><li>准备两个6厘米的菜，含有5毫升含0.1％BSA的PBS。上一碟板分离的细胞，使细胞至少5分钟解决。被异形的单细胞的性质在很大程度上取决于所用的细胞密度。例如，与荧光标记的GFP标记的细胞非常罕见，整个分离视网膜镀在一个6厘米的菜。对于较为丰富的细胞，细胞少，最初镀，使更容易收获的第一个微管细胞（或非常接近1）。 </li><li>将板倒置显微镜分离细胞。我们使用奥林巴斯IMT-2模型。使用微电极已制订一个科幻NE喷嘴（内径0.5毫米，外径1.04毫米）（ 图2），连同吸气管，收获一个单细胞。容易进入细胞，当它接近他们的菜放在微量。这是至关重要的吸气管是既不太长也不短距离倒置显微镜（我们用我们的倒置显微镜使用的吸气管为38.1厘米长）的阶段。如果管过长，细胞可能不被驱逐的微量有效地将收集管。我们使用旧的IMT-2型倒置显微镜的首要原因是，我们已经发现它具有理想的距离，吸气装配管阶段。后进入微管，细胞（或细胞）被驱逐到第二盘（洗板），以确保一个只有一个细胞基因表达谱的收获。在第二盘，它通常是必要的，要么删除一个新的微管外来细胞或利息细胞移动到一个不同的位置，以确保只有一个单细胞进入微管。 </li><li>当完全从邻近的细胞中分离单个细胞，使用一个新的微管吸的利益，在2.2节细胞，然后直接驱逐到0.2毫升PCR扩增含有4.5μL细胞裂解液管。细胞被驱逐到方管，小心不折断管微量的一角。样品可以在台式离心浸入细胞裂解液中纺简要。这纺通常完成后每5细胞，然后再在含有单细胞裂解液collection.Tubes年底应保持在冰上的单细胞分离时间。为了达到最佳效果，收集单细胞内分离后两个小时的窗口。在这个时候已经过去了，最好是进行逆转录步骤，因为更多的时间已经observ增加RNA降解的机会。 </li></ol> 3。反转录<ol><li>简单地旋转在台式minicentrifuge的样本，以确保所有的单个细胞，细胞裂解液中浸泡。促进细胞裂解，在70℃孵育90秒的样本。在热循环。立即放回冰管。 </li><li>离开管上冰2分钟。对于所有的试剂添加，使用过滤尖枪头，以防止污染。执行反转录，增加0.33微升三标（200 U /μL），0.05μLRNase抑制剂（40 U /μL），0.07μLT4基因32蛋白。孵育50分钟，在50°Ç在热循环反应混合物。为15分钟和地方冰灭活酶在70°C间。 </li><li>要删除自由的引子，添加我（20 U /μL）0.1μL酶切缓冲液（10X），0.8μL卫生署2 0（分子生物学级），0.1μL酶切。在37°C孵育30分钟1热循环。孵化在80°C反应25分钟，然后放在冰管的酶失活。 </li></ol> 4。尾矿和单细胞PCR <ol><li>尾矿的反应混合物加入6μl和使用热循环到20分钟，70℃孵育10分钟的样品在37°C，并保持在4°C间</li><li>加入10μl的PCR反应混合物的尾样本，并执行第二链的合成和PCR扩增，使用下列条件： <ol><li> 95℃2分钟</li><li> 37℃5分钟</li><li> 72℃16分钟</li><li> 93℃40秒</li><li> 67℃1分钟</li><li> 72℃6分钟，每个周期6秒</li><li>转到步骤4 34倍</li><li> 72℃10分钟</li><li>保持在4°C间</li></ol></li></ol> 5。 Affymetrix公司芯片的标签<ol><li>标签10-20微克的cDNA（浓度扩增的cDNA结果ING集团是从一个单细胞通常~~ 1微克/微升），Affymetrix公司的芯片获得了强大的杂交。首先，在80μL加入10-20μl到的单细胞cDNA 8μL1X一普所有的缓冲和稀释1μL（1点10分在1X一普所有缓冲区）DNaseI基因片段反应。在37°C孵育13分钟热循环灭活DNase I可在100°C和置于冰上15分钟。 </li><li>加入20μL5X TDT缓冲区，2.5μL生物素N6-ddATP（恩佐Biosciences公司），1μLTDT（在TDT缓冲区稀释1:8）。 </li><li>在37°C孵育90分钟，然后用5分钟在65°C储存在-20°C，或立即向Affymetrix的基因芯片杂交。使用标准的Affymetrix公司的协议，进行杂交。 </li></ol> 6。代表结果评估质量的cDNA，cDNA样本10μL1％琼脂糖凝胶装上。 cDNA的主要判断的尺寸范围（500-2000 BP）和强度的cDNA涂片（ 图1和图3a）。在图1所示的凝胶，每车道显示，从视网膜细胞的cDNA涂片分离E16.5。车道显示之间产生涂片，只有媒体时，被吸入进针，并存入PCR管。这些通道是从来没有完全缺乏淡淡涂抹，但他们没有表现出任何结果，特定基因的PCR引物被用来放大从他们的基因。此外，基因涂片强度可以有所不同（比较图1和图3a）。在图3A车道A，F，G和H包含强大的cDNA涂片检查，而车道B，C，D和E是相当少的健壮。 基因特异性PCR是用来作为一个从一个单细胞的cDNA质量二级屏幕。在图3的cDNA荧光视网膜神经节细胞的分离和他们睾丸D使用PCR引物对视网膜神经节细胞标记Brn3b和Pax6。对于这个实验中，1μL的cDNA进行PCR 30个循环。实时定量PCR技术将是一个最好的检测，但它可以是昂贵的，是没有必要的，仅仅是评估质量的cDNA。所有的单细胞8药检呈阳性了8 Brn3b和6 PAX6（ 图3b）。即使基因涂片Brn3b和Pax6带那么强劲。尽管这些PCR结果，这是我们的经验，如车道B，C，D和E的cDNA涂片不产生强大的杂交Affymetrix公司的阵列，一般可以避免。感光标记CRX基因特异性PCR被用来确定在单细胞的cDNA（ 图3b）的污染水平。选择一个感光标记，因为这些细胞使视网膜的大部分（〜70％），因此，任何细胞裂解发生的最有可能涉及棒photoreceptors。在这些基因的筹备工作，只有淡淡的CRX目前的金额，甚至后30 PCR循环。最后，这种基于PCR的屏幕可以用来确定一个特定的细胞类型的子集之前对他们进行全面的芯片分析得出的cDNA。这可以帮助优先细胞是异形的，因为这是在这个过程中最昂贵的一步。例如，我们已经确定了由视网膜神经节细胞亚群的筛选存在的，基因Tachykinin1（ 图3b）。 从PCR为基础的小屏幕，特别是单细胞的选择和一个Affymetrix的基因芯片杂交的cDNA。芯片的数据进行比较，可以使用标准的聚类算法发现的模式。如在图4的热图，生成以图形方式表示数据。有两个主要结论，对单细胞分析数据图4。首先，在特定的细胞类型的基因表达，发现在这些细胞类型几乎完全后进行单细胞协议。在大多数情况下的一种细胞类型的标记基因也被发现在第二个细胞类型，如观察，双极细胞表达一些“棒”的基因在较低水平，这在第二个单元格类型的表达式可以通过以下方式确认原位杂交或抗体染色。其次，在更多样化的无长突细胞和神经节细胞型材，基因表达的异质性是显而易见的。只表示，在约1/4的发展中国家的神经节细胞pou4f1，，而Nr4a2和Scube2是在不同的无长突细胞的异质性基因表达的两个例子。事实上，在热图中所示的基因只是一个小样本的数百个基因已被确定和发展的神经节细胞的标记，或标记为2,4无长突细胞的不同人群证实。 <p类“jove_content”&gt; <img alt="图1" src="/files/ftp_upload/3824/3824fig1.jpg" /> 图1。单细胞分析方法的流程图。第一视网膜是孤立和分离到单个细胞用木瓜蛋白酶。第二，单个细胞的收获与拉的玻璃针和存放到PCR管。裂解细胞的cDNA采用逆转录的PCR扩增。如所示的琼脂糖凝胶上产生的cDNA质量评估。车道后，在第一线的DNA梯状条带，从单个细胞扩增产物交替媒体控制（红色括号表示）的cDNA涂片。涂片质量（红色括号）Affymetrix的基因芯片杂交。 <img alt="图2" src="/files/ftp_upload/3824/3824fig2.jpg" /> 图2。针和吸气管组装的照片。单细胞分离使用的毛细作用拉玻璃微管（内径0.5毫米，外径1.04毫米）和吹吸气压力转移。重要的是，吸气管都足够长的时间，轻松地操纵和足够短，以可靠地履行单个细胞。毛细管拉成针的一个特写视图显示在B <img alt="图3" src="/files/ftp_upload/3824/3824fig3.jpg" /> 图3。与阴性对照组（A）的基因单细胞涂片检查和基因特异PCR的有代表性的例子。要确定的单细胞基因扩增后的质量，每个单细胞样本的10微升1％琼脂糖凝胶上运行。一抹黑，应该会出现500-2000 BP之间。额外的基于PCR的筛选特定基因可以帮助识别/确认被隔离的细胞类型和污染目前在样品量（B）。视网膜神经节细胞的特定引物标记Brn3b和Pax6进行了测试，以确认这些细胞（行1和2）的身份。评估准备感光污染量，为感光标记CRX的特定的引物被用来（第3行）。神经节细胞的亚群，还可以通过为，如Tachykinin1（4行）标记的筛选确定。 <img alt="图4" src="/files/ftp_upload/3824/3824fig4.jpg" /> 图4。单细胞标记基因的表达。选定在22个不同的单细胞表达的基因芯片结果显示在热图格式。从芯片信号的强度已经调整，以符合红色的强度。黑色表示没有信号的芯片。视网膜神经节细胞（研资局）的前体显示中分离出胚胎的时间点，而其他细胞分离成人视网膜。

<ol>
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U.S.A. 101, 1374-1379 (2004).</li><li>Chowers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+genes+preferentially+expressed+in+the+retina+using+a+custom+human+retina+cDNA+microarray.">Identification of novel genes preferentially expressed in the retina using a custom human retina cDNA microarray.</a> Invest. Ophthalmol. Vis. Sci. 44, 3732-3741 (2003).</li><li>Clarke, G. C., Leavitt, R. A., Andrews, B. R., Hayden, D. F., Lumsden, M. R., J, C., McInnes, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one-hit+model+of+cell+death+in+inherited+neuronal+degenerations.">A one-hit model of cell death in inherited neuronal degenerations.</a> Nature. 406, 195-199 (2000).</li><li>McEvoy, J. F. -. O., Zhang, J., Nemeth, J., Brennan, K., Bradley, R., Krafcik, C., Rodriguez-Galindo, F., Wilson, C., Xiong, M., Lozano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexpression+of+normally+incompatible+developmental+pathways+in+retinoblastoma+genesis.">Coexpression of normally incompatible developmental pathways in retinoblastoma genesis.</a> Cancer Cell. 20, 260-275 (2011).</li><li>Gelder, R. N. V. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Natl. Acad. Sci. U.S.A. 87, 1663-1667 (1990).</li><li>Ginsberg, S. D., Che, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profile+analysis+within+the+human+hippocampus:+comparison+of+CA1+and+CA3+pyramidal+neurons.">Expression profile analysis within the human hippocampus: comparison of CA1 and CA3 pyramidal neurons.</a> J. Comp. Neurol. 487, 107-118 (2005).</li><li>Iscove, N. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Representation+is+faithfully+preserved+in+global+cDNA+amplified+exponentially+from+sub-picogram+quantities+of+mRNA.">Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA.</a> Nat. Biotechnol. 20, 940-943 (2002).</li><li>Subkhankulova, T., Livesey, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+linear+and+exponential+amplification+techniques+for+expression+profiling+at+the+single-cell+level.">Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level.</a> Genome Biol. 7, R18 (2006).</li></ol>

没有利益冲突的声明。

被认为是考虑到他们的基因表达6,8更均匀的人口数量不断扩大的研究揭示强大的细胞，细胞变异。至少有一个实例，该基因表达的“噪音”已被证明发挥了重要的生物功能13。使用传统的全组织方法掩盖单个细胞之间的基因表达差异。这些实验产生一个“平均”的细胞，这可能不是代表14的表达谱。在这里，我们提出了一个从单一的视网膜细胞的基因表达模式的分离和鉴定...

Reaction mixtures:
Cell Lysis buffer
<table border="0">
	<tbody>
		<tr>
			<td>0.45 &mu;</td>
			<td>10X reaction buffer (100 mM Tris-HCl pH 8.3, 500 mM KCl, 2 mM MgCl2)</td>
		</tr>
		<tr>
			<td>0.23 &mu;l</td>
			<td>10% NP-40</td>
		</tr>
		<tr>
			<td>0.23 &mu;l</td>
			<td>0.1M DTT</td>
		</tr>
		<tr>
			<td>0.05 &mu;l</td>
			<td>RNase Inhibitor (40 U/&mu;l)</td>
		</tr>
		<tr>
			<td>0.05 &mu;l</td>
			<td>SUPERase-In (20U/&mu;l)</td>
		</tr>
		<tr>
			<td>0.13 &mu;l</td>
			<td>Modified Oligo d(T) primer 
			(TATAGAATTCGCGGCCGCTCGCGATTTTTTTTTTTTTTTTTTTTTTTT)</td>
		</tr>
		<tr>
			<td>0.09 &mu;l</td>
			<td>dNTPs (2.5 mM each)</td>
		</tr>
		<tr>
			<td>3.27 &mu;l</td>
			<td>dH20 (use molecular biology grade for all reaction mixtures)</td>
		</tr>
	</tbody>
</table>
Tailing Reaction Mixture
<table border="0">
	<tbody>
		<tr>
			<td>0.18 &mu;l</td>
			<td>100 mM dATP</td>
		</tr>
		<tr>
			<td>0.6 &mu;l</td>
			<td>10X reaction buffer(100 mM Tris-HCl pH 8.3, 500 mM KCl, 2 mM MgCl2)</td>
		</tr>
		<tr>
			<td>4.62 &mu;l</td>
			<td>dH2O</td>
		</tr>
		<tr>
			<td>0.3 &mu;l</td>
			<td>TdT (400 U/&mu;l)</td>
		</tr>
		<tr>
			<td>0.3 &mu;l</td>
			<td>RNase H (2 U/&mu;l)</td>
		</tr>
	</tbody>
</table>
PCR Reaction Mixture
<table border="0">
	<tbody>
		<tr>
			<td>10 &mu;l</td>
			<td>10x Ex-Taq HS Buffer with Mg2+</td>
		</tr>
		<tr>
			<td>10 &mu;l</td>
			<td>2.5 mM dNTPs</td>
		</tr>
		<tr>
			<td>0.2 &mu;l</td>
			<td>Modified Oligo d(T) primer (10 &mu;g/&mu;l)</td>
		</tr>
		<tr>
			<td>1 &mu;l</td>
			<td>Ex-Taq HS Polymerase (5U/&mu;l)</td>
		</tr>
		<tr>
			<td>68.8 &mu;l</td>
			<td>dH2O</td>
		</tr>
	</tbody>
</table>
10X One-Phor All Buffer
<table border="0">
	<tbody>
		<tr>
			<td>0.5M</td>
			<td>Potassium acetate</td>
		</tr>
		<tr>
			<td>0.1M</td>
			<td>Tris acetate (pH 7.6)</td>
		</tr>
		<tr>
			<td>0.1M</td>
			<td>Magnesium acetate</td>
		</tr>
	</tbody>
</table>
Solutions:
10X Phosphate buffered saline (1 liter)
80g NaCl 
2g KCl 
14.4 g Na2PO4 
2.4 g KH2PO4 
adjust to pH 7.4 with NaOH and bring the volume to 1 liter with water
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Vertical needle puller</td>
			<td>David Kopf Instruments</td>
			<td>Model 750</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microcapillary tubes</td>
			<td>Sigma</td>
			<td>P0674</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Aspirator tube assembly</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Corning filter tips (100-1000 &mu;l</td>
			<td>Fisher Scientific</td>
			<td>07-200-265</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hank's balanced salt solution (1X)</td>
			<td>Lonza BioWhittaker</td>
			<td>10-508F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEPES buffer (1M)</td>
			<td>MP Biomedicals</td>
			<td>091688449</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>04716728001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>papain</td>
			<td>Worthington</td>
			<td>LS03126</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III</td>
			<td>Invitrogen</td>
			<td>18080-044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase Inhibitor</td>
			<td>Applied Biosystems</td>
			<td>AM2682</td>
			<td>These two RNase inhibitors seem to work the best. Contaminants present in other inhibitors can contribute to a background smear.</td>
		</tr>
		<tr>
			<td>SUPERase-In</td>
			<td>Applied Biosystems</td>
			<td>AM2694</td>
			<td>These two RNase inhibitors seem to work the best. Contaminants present in other inhibitors can contribute to a background smear.</td>
		</tr>
		<tr>
			<td>Modified Oligo d(T) primer (gel purified)</td>
			<td>Oligos ETC</td>
			<td>&nbsp;</td>
			<td>We have used primers purchased from many different companies and have seen the most reliable and reproducible results from Oligos ETC.</td>
		</tr>
		<tr>
			<td>T4 gene 32 protein</td>
			<td>New England Biolabs</td>
			<td>M0300L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Exonuclease I</td>
			<td>New England Biolabs</td>
			<td>M0293S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TdT</td>
			<td>Roche</td>
			<td>3 333 574</td>
			<td>As with other reagents, using a TdT enzyme from other companies gave more variable results.</td>
		</tr>
		<tr>
			<td>RNase H</td>
			<td>Invitrogen</td>
			<td>18021-014</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ex-Taq HS Polymerase</td>
			<td>Takara</td>
			<td>RR006A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotin N6-ddATP</td>
			<td>Enzo Biosciences</td>
			<td>42809</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 1. Specific reagents and equipment.

single-cell profiling of developing and mature retinal neurons

高度专业化的，但非常小的细胞群体在许多组织中发挥的重要作用。细胞类型特异性标志物和基因表达程序极为罕见的细胞亚群的识别一直是一个挑战，使用标准的整个组织的方法。单个细胞的基因表达图谱允许获得了前所未有的细胞类型，包括只有一个很小的比例，共组织1-7。此外，这种技术可用于研究基因的表达，瞬时表达细胞小的数字，在动态发展过渡8的方案。 这种细胞的多样性的问题反复出现在中枢神经系统（CNS）神经连接可以相当不同的细胞之间发生9。不同的细胞类型的确切的数字是不准确不得而知，但据估计可能有多达1000个不同类型的皮质我tself 10。复杂的神经回路的功能（S）可能依靠一些罕见的神经类型和他们表达的基因。通过确定新的标志物和分子分类不同的神经元，单细胞的方法是在中枢神经系统的细胞类型分析特别有用。它也可能有助于阐明神经发育机制在神经祖发展的早期阶段，通过识别差异表达的基因和基因途径。 作为一种简单，方便地访问组织神经元具有相当的多样性，脊椎动物的视网膜是一个极好的模型系统研究细胞发育，神经细胞的分化和神经多样化的进程。然而，这种细胞的多样性，如中枢神经系统的其他部分，可以决定采取一种特定的细胞命运，推动视网膜祖细胞的遗传途径提出一个问题，特别是考虑到杆感光弥补马jority视网膜细胞总人口的11。在这里，我们报告单（ 图1）视网膜细胞中表达的转录的识别方法。单细胞分析技术允许在不同的细胞群体的视网膜2,4,5,12评估为异质目前的金额的。此外，这种方法揭示了一个新的候选基因，可能发挥的作用（S）在决策过程中的细胞命运发生在视网膜祖细胞亚群8。随着协议的一些简单的调整，这种技术可以用于许多不同的组织和细胞类型。

Watch this Scientific Journal Video about Single-cell Profiling of Developing and Mature Retinal Neurons at JoVE.com

Single-cell Profiling of Developing and Mature Retinal Neurons

描述一个孤立单一的视网膜细胞，随后他们的cDNA扩增的方法。单细胞转录揭示了在组织细胞的异质性目前的程度，并揭示了罕见的细胞群的新的标记基因。所附的协议，可以调整，以适应多种不同的细胞类型。

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

single-cell-profiling-of-developing-and-mature-retinal-neurons

Research

JoVE Journal

Neuroscience

14.1K 次观看.  Iowa State University. 描述一个孤立单一的视网膜细胞，随后他们的cDNA扩增的方法。单细胞转录揭示了在组织细胞的异质性目前的程度，并揭示了罕见的细胞群的新的标记基因。所附的协议，可以调整，以适应多种不同的细胞类型。

视频: Single-cell Profiling of Developing and Mature Retinal Neurons

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

从一个暗适应切片制备小鼠视网膜神经细胞的膜片钳录音

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

科研

神经科学

Profiling Voltage-gated Potassium Channel mRNA Expression in Nigral Neurons using Single-cell RT-PCR Techniques

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, difficult to separate and also not easily identified by their morphology. Thus, it is often difficult to analyze gene expression in a specific neuron type. Here we document a procedure that combines whole-cell patch clamp recording techniques with single-cell reverse transcription polymerase chain reaction (scRT-PCR) to profile mRNA expression in different types of neurons in the substantial nigra. Electrophysiological techniques are first used to record the neurophysiological and functional properties of individual neurons. Then, the cytoplasm of single electrophysiologically characterized nigral neurons is aspirated and subjected to scRT-PCR analysis to obtain mRNA expression profiles for neurotransmitter synthesis enzymes, receptors, and ion channels. The high selectivity and sensitivity make this method particularly useful when immunohistochemistry can not be used due to a lack of suitable antibody or low expression level of the protein. This method is also applicable to neurons in other brain areas.

Neurons are first characterized electrophysiologically. Then the cytoplasm from the recorded neuron is aspirated and subjected to reverse transcription-PCR analysis to detect the expression of mRNAs for neurotransmitter synthesis enzymes, ion channels, and receptors.

在使用单细胞RT - PCR技术的黑质神经元电压门控钾通道mRNA表达分析

In mammalian central nervous system, different types of neurons with diverse molecular and functional characteristics are intermingled with each other, ...

Cut-loading: A Useful Tool for Examining the Extent of Gap Junction Tracer Coupling Between Retinal Neurons

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic transmission. Increasing evidence indicates that gap junctions not only permit electrical current flow and synchronous activity between interconnected or coupled cells, but that the strength or effectiveness of electrical communication between coupled cells can be modulated to a great extent1,2. In addition, the large internal diameter (~1.2 nm) of many gap junction channels permits not only electric current flow, but also the diffusion of intracellular signaling molecules and small metabolites between interconnected cells, so that gap junctions may also mediate metabolic and chemical communication. The strength of gap junctional communication between neurons and its modulation by neurotransmitters and other factors can be studied by simultaneously electrically recording from coupled cells and by determining the extent of diffusion of tracer molecules, which are gap junction permeable, but not membrane permeable, following iontophoretic injection into single cells. However, these procedures can be extremely difficult to perform on neurons with small somata in intact neural tissue.
Numerous studies on electrical synapses and the modulation of electrical communication have been conducted in the vertebrate retina, since each of the five retinal neuron types is electrically connected by gap junctions3,4. Increasing evidence has shown that the circadian (24-hour) clock in the retina and changes in light stimulation regulate gap junction coupling3-8. For example, recent work has demonstrated that the retinal circadian clock decreases gap junction coupling between rod and cone photoreceptor cells during the day by increasing dopamine D2 receptor activation, and dramatically increases rod-cone coupling at night by reducing D2 receptor activation7,8. However, not only are these studies extremely difficult to perform on neurons with small somata in intact neural retinal tissue, but it can be difficult to adequately control the illumination conditions during the electrophysiological study of single retinal neurons to avoid light-induced changes in gap junction conductance.
Here, we present a straightforward method of determining the extent of gap junction tracer coupling between retinal neurons under different illumination conditions and at different times of the day and night. This cut-loading technique is a modification of scrape loading9-12, which is based on dye loading and diffusion through open gap junction channels. Scrape loading works well in cultured cells, but not in thick slices such as intact retinas. The cut-loading technique has been used to study photoreceptor coupling in intact fish and mammalian retinas7, 8,13, and can be used to study coupling between other retinal neurons, as described here.

An easy and convenient method to determine the extent of gap junction tracer coupling between retinal neurons is described. This technique enables one to investigate the function of the electrical synapses between neurons in the intact retina under different illumination conditions and at different times of the day and night.

剪切载荷：一个有用​​的工具，用于检查视网膜神经细胞之间的缝隙连接示踪剂耦合程度

In addition to chemical synaptic transmission, neurons that are connected by gap junctions can also communicate rapidly via electrical synaptic ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

同时光感受器和二阶神经元在两栖类动物的视网膜切片制备全细胞记录

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

记录光诱发神经元突触后反应在暗适应，小鼠视网膜切片准备利用膜片钳技术

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

成人脊髓细胞核在大规模平行单核 RNA 测序中的分离

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing platforms process all input cells indiscriminately. Sometimes, fluorescence-activated cell sorting (FACS) is used upstream to isolate a specifically labeled population of interest. A limitation of FACS is the need for high numbers of input cells with significantly labeled fractions, which is impractical for collecting and profiling rare or sparsely labeled neuron populations from the mouse brain. Here, we describe a method for manually collecting sparse fluorescently labeled single neurons from freshly dissociated mouse brain tissue. This process allows for capturing single-labeled neurons with high purity and subsequent integration with an in vitro transcription-based amplification protocol that preserves endogenous transcript ratios. We describe a double linear amplification method that uses unique molecule identifiers (UMIs) to generate individual mRNA counts. Two rounds of amplification results in a high degree of gene detection per single cell.

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-Seq

This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA sequencing.

荧光标记小鼠神经元的单细胞 RNA 序列的人工分选和双体外转录与绝对计数测序 (天后-Seq)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.

Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.

小鼠河马兴奋和类特异性抑制神经元的不同组织型切片培养的单细胞电位

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a ...