浙江省自然科学基金（LY15H310007），浙江省非营利技术应用研究项目（2016C37110），国家自然科学基金（U1503223,81673407），宁波国际科学和技术合作项目（2014D10019），宁波市生命科学与卫生创新队（2015C110026），广东省国际科技合作项目（2013B051000038），深圳基础研究计划（JCYJ20160331141459373），广东红香港技术合作资助计划（GHP / 012 / 16GD），香港研究资助局（15101014），香港理工大学（G-YBGQ）和宁波大学黄金大学基金。

所有程序均遵循国家卫生研究院（NIH）实验动物护理和使用指南（NIH出版物第80-23号，修订1996年），并得到宁波大学体育动物护理与使用委员会（IACUC）的批准（ SYXK-2008-0110）。 解决方案和文化媒体的准备注意:试剂和库存需要在无菌条件下制备。通过使用孔径为0.22μm的过滤器进行过滤灭菌。 <ol><li>通过向10mL双蒸水（ddH 2 O）中加入5mg的PLL制备聚-L-赖氨酸（PLL）储备溶液。过滤灭菌将PLL储存液等分并储存在-20°C几个月。 </li><li>通过将7.25g NaCl，0.4g KCl，0.14g NaH 2 PO 4，2.6g D-葡萄糖和5.94g 4-（2-羟乙基）-1-哌嗪乙磺酸加入到100mL的水中制备Krebs缓冲液ddH 2 &lt;/子&gt; 0。过滤灭菌将Krebs缓冲溶液在4℃下储存长达一个月。 </li><li>通过向100mL ddH 2 O中加入3.82g的Mg 2 SO 4来制备3.82％Mg 2 SO 4溶液。通过过滤灭菌。将Mg 2 SO 4溶液在4℃下储存数月。 </li><li>通过向100mL ddH 2 O中加入1.2g CaCl 2制备1.2％CaCl 2溶液。通过过滤灭菌。将CaCl 2溶液在4℃下储存数月。 </li><li>通过向100mL ddH 2 O中加入15克KCl来制备2M KCl溶液。通过过滤灭菌。将KCl溶液在4℃下储存数月。 </li><li>通过向100mL的ddH 2 O中加入1.8g D-葡萄糖来制备D-葡萄糖储备溶液。通过过滤灭菌。将D-葡萄糖溶液在4℃下等分并储存长达一个月。 </li><li>制备胞嘧啶β-D-阿拉伯呋喃糖苷（Ara-C）储备溶液通过将0.24g Ara-C加入到10mL ddH 2 O中通过过滤灭菌。将Ara-C储备溶液等分并储存在4℃下几个月。 </li><li>在Basal Medium Eagle（BME）中，使用25mL胎牛血清（FBS），2.5mL 100x谷氨酰胺，2.78mL 2M KCl溶液和2.5mL 100x抗生素制备250mL培养基（25-30只幼仔） 。将体积调节至250 mL，过滤灭菌。 </li><li>使用2.5 mL 100x谷氨酰胺，2.78 mL 2M KCl溶液和2.5 mL 100x抗生素在BME中制备25K培养基。将体积调节至250 mL，过滤灭菌。 </li><li>在BME中使用2.5 mL 100x谷氨酰胺和2.5 mL 100X抗生素制备5K培养基。将体积调节至250 mL，过滤灭菌。 注意:培养基，25K培养基和5K培养​​基需要新鲜准备</li><li>通过向1 mL丙酮中加入5 mg FDA来准备FDA储备溶液。将FDA储存溶液储存在4°C长期储存/ LI&gt; <li>通过在1mL ddH 2 O中加入1mg PI制备PI储备溶液。将PI储备溶液在4℃下储存几个月。 </li><li>通过向1mL ddH 2 O中加入5mg Hoechst 33342制备Hoechst储备溶液。将Hoechst储备溶液在4℃下储存几个月。 </li></ol> 2.解剖解决方案的准备注意:在解剖前1 d做。 <ol><li>通过加入15 mL Krebs缓冲液，1.2 mL 3.82％Mg 2 SO 4溶液和0.45 g牛血清白蛋白（BSA），加入到150 mL ddH 2 O中来制备解剖溶液。用NaOH调节pH至7.4。 </li><li>标记5×50mL管，如下:1,2,3,4和5。 </li><li>将40 mL解剖溶液放入带过滤器的50mL注射器中。将30mL的解剖溶液过滤到管1中，并将2mL分别加到几个35mm的细胞培养皿中，这些培养皿将用于处理组织。 </li><li>要tub 2，在50mL的解剖溶液中加入12.5mg胰蛋白酶。通过涡旋完全混合。过滤灭菌</li><li>向管3中加入1.2mg DNAse I，7.8mg大豆胰蛋白酶抑制剂和150μL3.82％Mg 2 SO 4溶液于15mL解剖溶液中。通过涡旋完全混合。过滤灭菌</li><li>对管4，从管3中加入5mL溶液。加入10.5mL解剖溶液。过滤灭菌</li><li>向管5中加入100μL3.82％Mg 2 SO 4溶液和15μL1.2％CaCl 2溶液在12.5mL的解剖溶液中。通过涡旋完全混合。过滤灭菌</li><li>使用前将所有的管保存在4°C。 </li></ol> 3.涂覆细胞培养板注意:在解剖前1 d做。 <ol><li>加入1 mL PLL储备溶液至100mL ddH 2 O（终浓度:5μg/ mL）。大衣塑料通过加入5μg/ mL聚-L-赖氨酸溶液，6孔或12孔细胞培养板（6孔细胞培养板每孔2mL或12孔细胞培养板每孔1mL）。 </li><li>在室温下孵育1天（或37℃，2小时）。解剖前1-2 h，用移液管取出溶液。用ddH 2 O洗涤一次，在细胞培养罩中干燥。 </li></ol> 4.加工8日龄Sprague-Dawley大鼠的组织。 <ol><li>把一个100毫米的盘子放在冰上。使用一把无菌剪刀将大鼠幼仔放入盘中。 </li><li>将剪刀插入孔中，将头骨的两侧从耳朵切割到眼睛。用镊子提起头骨。确保整个大脑在头骨中。使用一对镊子将小脑隔离并放入上述35mm的盘中。 </li><li>在解剖显微镜下工作。用两对镊子去除脑膜和血管。 </li><li>用刀片切碎纸巾。将组织放入管1中。在室温和1500×g离心5分钟。 </li><li>吸出上清液。保存颗粒。 </li><li>将管2中的溶液加入沉淀。轻轻晃动重悬。 </li><li>将管置于37℃水浴中15分钟。每3分钟轻轻晃动一次。 </li><li>将管4中的溶液加入到该溶液中。摇匀并离心5分钟，室温和1500 x g。 </li><li>吸出上清液。保存颗粒。 </li><li>将溶液加入管3中以重悬。 </li><li>准备两个15 mL管。将一半的细胞放在每个管中。使用棉塞巴斯德移液器将60-70x上下移动，使细胞匀浆。 </li><li>在管5中加入3 mL溶液至每个15 mL管中。让他们在室温下静置10分钟，然后用棉塞巴斯德吸管小心地去除上清液。将上清液放入新的15 mL管中，并在室温和1500 x g离心5 min。 </li><li>一个旋转上清液。保存颗粒。 </li><li>将培养基添加到沉淀中以获得约1.5×10 6个细胞/ mL的细胞密度（10只幼仔约100mL）。 </li><li>将细胞置于培养板中，并在37℃和5％CO 2的培养箱中培养。 </li></ol> 5.培养过程中加入Ara-C和D-葡萄糖<ol><li> 24小时后，加入Ara-C储备溶液（12孔细胞培养板10μL/孔或6孔细胞培养板20μL/孔;终浓度:1 mM），以抑制胶质细胞的生长。 </li><li>在第7天，对于12孔细胞培养板，每孔加入50μLD-葡萄糖储备溶液或每孔100μL用于6孔细胞培养板，使得终浓度为1mM。 </li></ol> 6.GC-PI双重染色和CGI文化中的FDA-PI-Hoechst三重染色<ol><li> 通过加入20μL的FDA储备溶液（准备FDA-PI工作溶液）最终浓度:10μg/ mL）和50μLPI储备溶液（终浓度:50μg/ mL）在10mL PBS中的溶液。通过涡旋混合并将其置于冰上。 <ol><li>对于FDA-PI-Hoechst工作溶液，加入20μLFDA储备溶液（终浓度:10μg/ mL），50μLPI储备溶液（终浓度:50μg/ mL）和10μLHoechst储备溶液（终浓度:5μg/ mL）在10mL的PBS中。通过涡旋混合并将其置于冰上。 注意:在使用之前，新鲜准备FDA-PI工作解决方案和FDA-PI-Hoechst工作解决方案。 </li></ol></li><li>将细胞培养板从培养箱中取出。放在冰上</li><li>吸取培养基并用冷PBS代替。 注意:解决方案的变化必须缓慢而小心地进行。避免用移液器吸头接触细胞。 </li><li>吸入冷PBS，并用冷的FDA-PI或FDA-PI-Hoechst工作溶液（500μL/我们）进行更换12孔细胞培养板或1mL /孔用于6孔细胞培养板）。在冰上离开5分钟。 </li><li>吸取FDA-PI或FDA-PI-Hoechst工作溶液，加入冷PBS（12孔细胞培养板100μL/孔或6孔细胞培养板50μL/孔）。 注意:摄影时不应让细胞干燥。 </li><li>使用荧光显微镜拍摄图像。使用通过波长为450 - 490 nm的激发滤光片。分别在520,620和460nm检测FDA，PI和Hoechst的荧光发射。在相同条件下，使用荧光显微镜的相位对比模式在正常光照下拍摄图像。 注意:在FDA-PI或FDA-PI-Hoechst染色后15分钟内拍摄图像。曝光时间为100-300 ms，模拟增益为2.8x。 </li></ol> 7.神经元生存能力评估<ol><li> 对于FDA-PI双染色，覆盖过滤后检测到的细胞的图像通过在图形编辑器软件中通过在PI阳性层上拖动FDA阳性层，通过不同的荧光滤光片。 <ol><li>通过在"图层"面板的"不透明度"字段中输入"50％"来调整FDA阳性层的不透明度。通过单击"图层"菜单中的"合并可见"按钮合并两个图层。通过在"图像"|下的"对比度"字段中输入"50"来设置重叠图像的对比度"调整"| "亮度/对比度"。确保覆盖图像中没有FDA和PI双阳性细胞。 </li></ol></li><li> 对于FDA-PI-Hoechst三重染色，通过在图形编辑器软件中拖动PI阳性层上的FDA阳性层，在过滤不同的荧光过滤器之后，覆盖检测到的细胞的图像。通过在"图层＆＃"中的"不透明度"字段中输入"50％"来调整FDA阳性层的不透明度，34;面板。 <ol><li>通过单击"图层"菜单中的"合并可见"按钮合并两个图层。拖动合并图层上的Hoechst正层。通过在"图层"面板的"不透明度"字段中输入"50％"来调整Hoechst正层的不透明度。通过单击"图层"菜单中的"合并可见"按钮合并两个图层。 </li></ol></li><li>通过比较在荧光模式下拍摄的图像与在相位对比模式下拍摄的图像，可以将CGNs中的大型不规则胶质细胞视觉辨认为直径比CGN大三倍的细胞被认为是胶质细胞，应该被排除。 </li><li> 通过在图像处理程序（ 例如， ImageJ） 4中使用细胞计数器插件分别计数可行神经元和死神经元的数量。 <ol><li>对于FDA-PI双重染色，手动标记小的FDA阳性细胞可行的神经元。手动将PI阳性细胞标记为死神经元。对于FDA-PI-Hoechst三重染色，手动将小型的FDA和Hoechst-双阳性细胞标记为可行的神经元。手动将PI-和Hoechst-双阳性细胞标记为死亡神经元。 </li></ol></li><li>通过使用以下等式计算神经元活力的百分比:神经元存活力的百分比= [活神经元数量（死亡神经元数+可行神经元数）]×100％。对于每个井，选择五个随机场，平均神经元生存率的百分比。 </li></ol>

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作者没有什么可以披露的。

该协议是从先前已经描述过的程序6,7修改的。研究人员花费时间尝试通过在体外培养原代神经元时改善条件来减少非神经元细胞生长8 。然而，即使改善的培养条件，一些胶质细胞仍然存在。此外，非神经元细胞在原代神经元培养中是必需的，因为它们有助于神经元生长和成熟9 。在这项研究中，Ara-C被用来尽可能减少神经胶质细胞的生长，尽管在CGN培养物中存在约1-5％的神经胶质细胞。其他组的研究也提出了这个问题10 。在CGN文化中，胶质细胞的大小和形状与神经元大不相同。 CGN的直径约为10μm，成熟的CGN具有连接神经元的丰富过程。豪ver，培养物中胶质细胞的直径相对较大，约为30-100μm，因此，图像中的神经元细胞与胶质细胞的分离很容易。因此，FDA-PI和FDA-PI-Hoechst染色的一个优点是准确测量原代神经元培养物中的神经元活力，其中非神经元细胞生长不被培养技术阻止。 低浓度钾诱导的细胞凋亡CGN培养，25K培养基处理细胞作为对照，代表了研究神经细胞凋亡的一个很好的模型。许多组织，包括我们的实验室，都使用这个经典模型研究神经元2,11的潜在凋亡机制。因此，该模型用于诱导神经元死亡。本方案中使用的染料浓度和染色持续时间已被优化。低浓度的染料和短的染色持续时间可能导致染色不足，导致不准确的couning死神经元。然而，高浓度的染料和延长的染色持续时间可进一步诱导神经元死亡，并在细胞活力的估计中引起偏见。因此，添加染料后，应尽快拍摄图像。在FDA-PI双染色图像中，小的FDA阳性细胞被标记为可行的神经元。然而，细胞体趋向于在CGN文化中组合起来。因此，通过FDA染色可能难以分离活细胞。在这项研究中，Hoechst是一种核染色，用于提高准确度。在FDA-PI-Hoechst三重染色图像中，具有FDA阳性细胞体和Hoechst阳性细胞核的小细胞可以被认为是可行的神经元。 与比色细胞毒性测定相比，FDA-PI和FDA-PI-Hoechst染色需要更多时间来评估CGN培养中的神经元活力。因此，目前的方案可能不适合筛选神经保护药物。但是，这个限制可以通过使用高内容成像系统来解决。这种技术的另一个限制是，CGN和胶质细胞不能被PI区分开来。然而，因为神经元比神经胶质细胞更容易受神经毒素的影响，PI阳性死细胞主要是神经元12 。 除了FDA，PI和Hoechst之外，其他染料如MTT，SYTO13和SYBR14也用于测量细胞活力13,14 。对于MTT测定，对神经毒素不敏感的神经胶质细胞可以将MTT转化成甲，，导致神经元活力的过度估计。 SYTO13是细胞可渗透的，当与DNA或RNA结合时具有高的绿色荧光产率。以前的研究表明，SYTO13染色主要表现在CGN培养物中的凋亡细胞，但不区分神经胶质细胞13 。 SYBR14是一种膜永久性核酸染料。 SYBR14-PI双染是我们的以测量细胞活力，因为两种染料标记DNA，从而规避了模糊度14 。然而，SYBR14-PI双染不能在CGN培养中有效区分神经元的神经胶质细胞。因此，可以区分胶质细胞与神经元的FDA-PI和FDA-PI-Hoechst染色有利于准确评估CGN培养中的神经元活力。 最后，FDA-PI和FDA-PI-Hoechst染色不限于CGN培养物中神经元活力的分析。许多混合细胞培养物（ 例如，初级皮层培养物和原代海马培养物）也含有神经元和神经胶质细胞。因此，类似的策略可以应用于这些混合细胞培养物，以准确分析神经元的活力。

通常使用比色细胞毒性测定法（例如3-（4,5-二甲基-2-噻唑基）-2,5-二苯基-2-H-四唑溴化物（MTT）测定法来测量体外细胞活力。来自大鼠的原代培养的小脑颗粒神经元（CGN）对各种神经毒素敏感，包括1-甲基-4-苯基吡啶鎓离子，过氧化氢和谷氨酸盐1,2 。因此，CGN培养可用作神经科学领域的体外模型。 CGN培养物可以含有多种细胞，包括神经元和神经胶质细胞，其可占CGN培养物中总细胞的约1％。然而，与神经元相比，神经胶质细胞对神经毒素的反应不同，导致通过比色测定法测量的神经元存活力的偏差3 。 在活细胞中，荧光素二乙酸酯（FDA）可以通过酯酶转化为荧光素。碘化丙啶（PI）可以在穿透死细胞后与DNA相互作用，可用于指示培养物中的凋亡。因此，FDA-PI双染可以同时评估活细胞和死细胞，这表明通过组合比色法可以更精确地测量细胞活力。此外，通过添加Hoechst蓝色荧光染色剂可以进一步提高细胞活力的准确性。本文提出的方案描述了FDA-PI双染色和FDA-PI-Hoechst三重染色，可用于准确分析原代培养的CGNs中的神经元活力。 该协议利用其不同尺寸和形状可视化和区分CGN和胶质细胞。染色后，从通过荧光显微镜拍摄的代表性图像中计数活神经元和死亡神经元的数量。通过比较典型的CGN t来排除大尺寸胶质细胞在荧光模式下，在相位对比模式下拍摄。可以进行类似的策略来测量含有神经元和神经胶质细胞的混合细胞培养物中的神经元活力，例如原代皮层培养物和海马培养物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma</td>
			<td>P2636</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-Arabinofuranoside</td>
			<td>Sigma</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10099141&#160;</td>
			<td>high quality FBS is essential for culture</td>
		</tr>
		<tr>
			<td>100&#215; glutamine</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>100&#215; anti-biotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td></td>
		</tr>
		<tr>
			<td>BME medium</td>
			<td>Gibco</td>
			<td>21010046&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein diacetate</td>
			<td>Sigma</td>
			<td>F7378</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Yesen</td>
			<td>40731ES10</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit Anti-GAP43 antibody</td>
			<td>Abcam</td>
			<td>ab75810</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse Anti-GFAP antibody</td>
			<td>Cellsignaling</td>
			<td>3670</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sangon Biotech</td>
			<td>A602440</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Sigma</td>
			<td>T4665</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse&#160;</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Volac</td>
			<td>Z310727</td>
			<td>burn&#160; the tip round before use</td>
		</tr>
		<tr>
			<td>12-well cell culture plates</td>
			<td>TPP</td>
			<td>Z707783</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>TPP</td>
			<td>Z707759</td>
			<td>high quality cell culture plate&#160; is essential for culture</td>
		</tr>
		<tr>
			<td>Filter</td>
			<td>Millipore</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 5 ml</td>
			<td>Excell Bio</td>
			<td>CS017-0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet 10 ml</td>
			<td>Excell Bio</td>
			<td>CS017-0004</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissect microscope</td>
			<td>Shanghai Caikang</td>
			<td>XTL2400</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific&#160;</td>
			<td>311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope&#160;</td>
			<td>Nikon</td>
			<td>TI-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence filter and emmision cubes</td>
			<td>Nikon</td>
			<td>B-2A, G-2A, UV-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo software</td>
			<td>Nikon</td>
			<td>NIS-Elements</td>
			<td></td>
		</tr>
		<tr>
			<td>Graphics editor softeware</td>
			<td>Adobe</td>
			<td>Photoshop CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Image process softeware</td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of neuronal viability using fluorescein diacetate-propidium iodide double staining in cerebellar granule neuron culture

原代培养的小脑颗粒神经元（CGNs）已广泛用作神经科学和神经药理学研究中的体外模型。然而，CGN培养中神经胶质细胞和神经元的共存可能导致精确评估神经元活力的偏差。荧光素二乙酸酯（FDA）和碘化丙啶（PI）双染法已被用于通过同时评估活细胞和死细胞来测量细胞活力。我们使用FDA-PI双染来提高比色测定的敏感性，并评估CGNs中的神经元活力。此外，我们添加蓝色荧光DNA污渍（ 例如 Hoechst）以提高准确性。该协议描述了如何通过在CGN培养中使用这些方法来提高神经元活力评估的准确性。使用该方案，可以通过使用荧光显微镜来排除胶质细胞的数量。可以应用类似的策略来区分不想要的gli来自各种混合细胞培养物中的神经元的细胞，例如原代皮质培养物和海马培养物。

GAP43（红色）和GFAP（绿色）的双重免疫染色分别用于分析神经元和神经胶质细胞的形状。神经元和神经胶质细胞均存在于CGN培养物中。 GFAP阳性神经胶质细胞大，形状不规则，如图像中的箭头所示（ 图1 ）。当用于测量神经元活力时，细胞活力的传统测定法不能区分神经元神经元。因此，可以区分胶质细胞与神经元的FDA-PI和FDA-PI-Hoechst染色有利于准确评估CGN培养中的神经元活力。 低钾攻击用于诱导CGN培养中的神经元死亡。代表性的图像显示用低钾培养基（5K），正常培养基（25K）或起源通过FDA-PI双染（ 图2A ）或FDA-PI-Hoechst三重染色（ 图2B ）进行分析。通过各种方法测量的神经元活力示于图3 （平均值±SEM）。对照组，25K组和5K组，通过FDA-PI双染测定细胞活力分别为99.8±4.2％，98.2±2.9％和43.9±8.6％（ 图3A ）。对照组，25K组和5K组，通过FDA-PI-Hoechst三重染色测定细胞活力分别为99.8±1.6％，96.7±4.4％和48.3±4.4％（ 图3B ）。对照组，25K组和5K组MTT测定细胞活力分别为102.1±3.9％，96.5±1.7％，57.5±5.7％（ 图3C ）。对照组，25K组和5K组乳酸脱氢酶（LDH）释放百分比分别为100.0±5.5％，94.5±11.2％和202.1±15.3％ <strong&gt;图3D）。神经元生存能力不能通过LDH测定直接计算2 。 MTT测定评估NADPH依赖性细胞氧化还原酶的活性，其反映活细胞数量2 。然而，当用于测量CGN培养中的神经元活力时，该方法不能区分神经元的神经胶质细胞。通过使用FDA-PI和FDA-PI-Hoechst染色，可以排除胶质细胞的数量，并可以准确测量神经元的活力。此外，通过FDA-PI或FDA-PI-Hoechst染色测量的5K培养基处理的CGN中的神经元活力略低于通过MTT测定测量的神经元存活力。这可能是因为大多数对5K中度诱导的神经毒性不敏感的神经胶质细胞被FDA-PI或FDA-PI-Hoechst染色排除，而不是通过MTT测定法排除。 <img alt="图1" src="/files/ftp_upload/55442/55442fig1.jpg" /><br/&gt; 图1: CGN文化中都存在小神经元和大型不规则胶质细胞。在第8天，CGNs分别用GAP43（红色）和GFAP（绿色）进行双重免疫染色，用于神经元和神经胶质细胞。相位图显示细胞的形态。当用于测量神经元活力时，细胞活力的传统测定法不能区分神经元神经元。因此，可以区分胶质细胞与神经元的FDA-PI和FDA-PI-Hoechst染色有利于准确评估CGN培养中的神经元活力。刻度棒:100μm。蓝色箭头:典型神经元;白色箭头:典型的胶质细胞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55442/55442fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55442/55442fig2.jpg" /> <s图2: FDA-PI双重染色和FDA-PI-Hoechst三重染色显示CGN培养中低钾诱导的神经元死亡。在第8天，将CGN培养物切换至5K或25K培养基。对照培养中的培养基没有改变。攻击24小时后，通过（ A ）FDA-PI双染色或（ B ）FDA-PI-Hoechst三重染色测定CGN培养物。刻度棒=100μm。箭头:典型的胶质细胞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55442/55442fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55442/55442fig3.jpg" /> 图3: CGN培养中神经元活力的定量。在第8天，将CGN培养物切换至5K或25K培养基。控制文化中的媒介没有改变。在攻击24小时后，通过（ A ）FDA-PI双染色，（ B ）FDA-PI-Hoechst三重染色和（ C ）MTT测定分析细胞活力。 （ D ）通过LDH测定分析LDH释放。数据表示为均值±SEM。 ** p &lt;0.01与对照组（ANOVA，Tukey&#39;s检验）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55442/55442fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture at JoVE.com

Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

该协议描述了如何使用荧光素二乙酸酯（FDA）和碘化丙啶（PI）双重染色在培养的小脑颗粒神经元中准确测量神经元活力，这是一种初级神经元培养物，用作神经科学和神经药理学研究中的体外模型。

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

Primary cultured Cerebellar Granule Neurons (CGNs) have been widely used as an in vitro model in neuroscience and neuropharmacology research. However, the ...

assessment-neuronal-viability-using-fluorescein-diacetate-propidium

Nanyang Technological University

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Neuroscience

11.6K 次观看.  Ningbo University. 该协议描述了如何使用荧光素二乙酸酯（FDA）和碘化丙啶（PI）双重染色在培养的小脑颗粒神经元中准确测量神经元活力，这是一种初级神经元培养物，用作神经科学和神经药理学研究中的体外模型。 

视频: Assessment of Neuronal Viability Using Fluorescein Diacetate-Propidium Iodide Double Staining in Cerebellar Granule Neuron Culture

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

科研

神经科学

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

小胶质细胞的小脑颗粒细胞使用L-亮氨酸甲酯选择性耗尽

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

低密度原海马神经元文化

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their post-natal development, abundance, and accessibility, CGNs are an ideal model to study neuronal processes, including neuronal development, neuronal migration, and physiological neuronal activity stimulation. In addition, CGN cultures provide an excellent model for studying different modes of cell death including excitotoxicity and apoptosis. Within a week in culture, CGNs express N-methyl-D-aspartate (NMDA) receptors, a specific ionotropic glutamate receptor with many critical functions in neuronal health and disease. The addition of low concentrations of NMDA in conjunction with membrane depolarization to rodent primary CGN cultures has been used to model physiological neuronal activity stimulation while the addition of high concentrations of NMDA can be employed to model excitotoxic neuronal injury. Here, a method of isolation and culturing of CGNs from 6 day old pups as well as genetic manipulation of CGNs by adenoviruses and lentiviruses are described. We also present optimized protocols on how to stimulate NMDA-induced excitotoxicity, low-potassium-induced apoptosis, oxidative stress and DNA damage following transduction of these neurons.

This protocol describes a simple method for isolating and culturing primary mouse cerebral granule neurons (CGNs) from 6-7 day old pups, efficient transduction of CGNs for loss and gain of function studies, and modelling NMDA-induced neuronal excitotoxicity, low-potassium-induced cell death, DNA-damage, and oxidative stress using the same culture model.

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

摘要

探索更多视频

此视频中的章节

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

谷氨酸和GABA快速微离子导入:一个有用的工具，调查突触整合

小脑颗粒神经元的遗传操作

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

小胶质细胞的小脑颗粒细胞使用L-亮氨酸甲酯选择性耗尽

低密度原海马神经元文化

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

一个简单的神经元机械损伤方法学研究

使用荧光素二乙酸酯 - 碘化丙啶双重染色在小脑颗粒神经元培养中评估神经元活力

摘要

探索更多视频

此视频中的章节

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

神经元对神经胶质细胞在微流控文化互动平台（MCP）的神经元轴突和神经胶质细胞共培养系统的影像学分析

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

小脑颗粒神经元的遗传操作

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

小胶质细胞的小脑颗粒细胞使用L-亮氨酸甲酯选择性耗尽

低密度原海马神经元文化

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

一个简单的神经元机械损伤方法学研究

谷氨酸和GABA快速微离子导入:一个有用的工具，调查突触整合