我们要感谢 Mansky 实验室提供极速冻设备。这项工作的一部分是在明尼苏达大学的表征设施中进行的，该设施通过材料研究科学与工程中心（MRSEC;奖项编号 DMR-2011401） 和国家神经科学课程倡议 （NNCI;奖项编号 ECCS-2025124） 计划。我们要感谢病毒环境中的HIV行为中心（B-HIVE;1U54AI170855-01）和杜克大学HIV结构生物学中心（DCHSB;U54AI170752） 中心。

1. 电网准备
注:在实验设计中，计划每次浸入冷冻期间最多 8-12 个网格，每孔最多 4-5 个网格。超过此值将导致非常长的冷冻过程，这可能会导致细胞压力增加、冰污染和用户错误。
<ol><li>辉光放电<ol><li>在辉光放电装置中，用穿孔碳支撑膜加载适当数量的网格，以 15 mA 的电流在 0.39 mBar 气氛下以 0.39 毫秒的电流进行辉光放电。</li>
		<li>确保电网的碳面朝上。完成后，将网格转移到衬有滤纸的干净容器中（图1A1）。 注意:只要材料对电池无毒（不包括任何铜网格），就可以使用其他网格。Finder 网格对于关联研究非常有用。也可以使用其他材料制成的网格。许多实验室在 SiO2 支持网格 8,9 上生长细胞取得了良好的结果。</li>
	</ol></li>
	<li>依从性治疗<ol><li>将网格、纤连蛋白、PBS、板和镊子转移到生物安全柜中。通过在无菌表面（例如 6 孔板的盖子）上微量移液两个大大小的贴壁溶液点，用 20 μg/mL 牛纤连蛋白处理网格的两侧。确保点足够大以包裹整个网格（建议约 100 μL）。 注:在用选定的贴壁溶液处理之前，可以选择在此处对网格进行光微图案化，以优化网格正方形中心细胞的附着。降低单元格附着到条形图的能力将增加可成像单元格的数量。这是涉及冷冻FIB研磨的实验的理想选择。</li>
		<li>使用 5/15 镊子操作网格。确保在溶液中处理网格的两侧。 注意:可以使用其他粘合剂溶液（纤维蛋白原、胶原蛋白、其他细胞外基质成分等）。这里使用纤连蛋白，因为它在多种细胞系中都能获得良好的结果。（U2OS、HeLa、Vero、Calu-3、Tzm-bl）。5/15 镊子非常有用，因为它的几何形状可以提高 6 孔板、35 mm 培养皿和微型载玻片周围的可操作性。这样可以减少网格上的机械应力，并形成更完整的碳膜。确保在开始之前将所有必需的材料都放在生物安全柜中，因为这样可以减少网格污染的可能性。</li>
	</ol></li>
	<li>附加网格<ol><li>一旦用牛纤连蛋白处理，网格就会变得粘稠。使用 5/15 镊子，将网格的非碳表面轻轻接触空盘/盘子的底部，然后打开镊子。网格应易于附着在新表面上（图1A2）。 注意: 尽量避免将网格放在盘子/盘子的直接中心或边缘。添加细胞时，细胞密度有在板中心积聚的趋势。这可能导致网格具有过高的细胞密度。或者，这里可以使用3D打印的网格支架，而不是将细胞直接连接到培养皿/板12的表面。</li>
	</ol></li>
	<li>孵化<ol><li>将网格在纤连蛋白贴壁溶液（20μg/ mL）中孵育30分钟。为确保网格在此过程中不会变干，每15分钟将1-2滴更粘附的溶液直接滴在网格顶部（图1A3）。 注意:孵育期将根据所使用的贴壁溶液而有所不同。对于牛纤连蛋白，推荐时间为 30 分钟。</li>
	</ol></li>
	<li>洗涤<ol><li>孵育期过后，通过微量移液去除多余的粘附溶液。通过将PBS滴微量移液液直接在网格顶部来清洗网格。重复此操作 2-3 次。（图 1A4）。</li>
	</ol></li>
	<li>灭菌<ol><li>使用紫外线对生物安全柜中的网格进行消毒1小时。将网格放置在尽可能靠近紫外线源的位置，以最大限度地提高灭菌效果（图1A5）。为确保网格在此过程中不会变干，每 10 分钟将 1-2 滴 PBS 直接滴在网格顶部。 注意:步骤 1.4-1.6 可以同时执行。</li>
	</ol></li>
</ol>2. 播种网格
<ol><li>细胞计数:<ol><li>使用机械或酶法分离和计数细胞。在计数之前确定用于接种的最佳细胞数。对于接种在 6 孔板中的 U2OS，每孔使用 6 x 104 至 1.6 x 105 个细胞之间的任意位置（图 1B1）。 注:接种的细胞数量将根据细胞类型、孔的表面积、使用的培养皿或微载玻片、接种和浸入冷冻之间的时间以及实验设计而变化。测试一系列细胞数量，以确定在骤降冻结时将导致每个网格方块产生 0.25-1 个细胞的条件。需要注意的是，高细胞密度会降低传热速度，并可能影响玻璃化过程。为了解决这个问题，一些实验室已经发现使用细胞过滤器取得了成功，这种过滤器的作用是防止细胞团块形成13。最后，该协议导致细胞随机附着在碳层上。如果需要有针对性的附件，可以使用光微图案化（参见步骤 1.3）14。</li>
	</ol></li>
	<li>添加细胞和生长培养基:通过微量移液管将细胞添加到孔中，避免气泡（图1B2）。在 6 孔板中，建议总孔体积为 1.5-2.0 mL。 注意: 建议在添加整个体积之前小心地弄湿网格周围。这将有助于防止网格在溶液中分离和浮动。轻轻地左右移动板，以促进孔上的细胞均匀分布。不要以圆周运动旋转板，因为这会导致孔中心分配过多的细胞密度。</li>
	<li>孵育:根据实验设计，将载玻片在37°C下孵育适当的时间，这将取决于下游应用。对于本例（HIV分子克隆的转染），孵育16-24小时（图1B3）。 注意:较长的孵育时间将导致更多的细胞分裂周期。因此，请相应地调整接种的细胞起始数量。</li>
</ol>3. 转染
<ol><li>制备转染混合物:<ol><li>制备适当的阳离子脂质体试剂，用于将质粒转染到所选细胞系中。</li>
		<li>在该转染示例中，每孔（在 6 孔板中）使用 1 μg 总质粒 DNA，以 1:3 的比例转染 HIViΔEnv 质粒与 psPAX2 质粒。对于每个孔，将 1 μg DNA 稀释到 50 μL 无血清 DMEM 中。通过反复微量移液或轻轻旋转板来匀浆混合物。</li>
		<li>对于每个孔，在无血清DMEM中稀释3μL转染试剂，然后通过微量移液或轻轻旋转再次匀浆混合物。将整个稀释的转染试剂混合物加入稀释的DNA混合物中（步骤3.1.2），并在室温（RT）下孵育15分钟（图1C1）。 注意:当该混合物孵育时，用 1.5 mL 新鲜 DMEM 替换孔中的培养基。小心不要将格栅从井底移开。</li>
	</ol></li>
	<li>为了应用转染混合物，将步骤3.1.3中的100μL混合物逐滴移液到每个孔中，将液滴集中在网格上以确保接触（图1C2）。转染后16-24小时更换生长培养基（图1C3）。</li>
</ol>4.骤降冻结
注意:将细胞保持在37°C，直到需要印迹。此外，在整个过程中，请务必记下电网的碳侧。
<ol><li>细胞密度<ol><li>在倒置光学显微镜中检查网格，并注意每个网格上的细胞密度。 注意:每个网格方块少于 0.25 个单元格（或每 4 个网格方块一个单元格）的网格被视为"空"。每个网格方格有 0.25-1 个像元的网格被认为是"正常"的。每个格子方块具有 1 个以上像元的格网被视为"已满"。</li>
		<li>记下每个网格上的细胞密度，以便在速冻期间稍后调整印迹时间。将2-3mL PBS加入空板/培养皿中，并加热至37°C。</li>
	</ol></li>
	<li>准备基准点<ol><li>为了制备用于下游冷冻断层扫描应用的金基准点，将 50 μL 10 nm 胶体金珠溶液移液到干净的 1.5 mL 试管中。向试管中加入 2 μL 1 mg/mL BSA，并通过反复微量移液匀浆。</li>
		<li>将试管以15,000-20,000× g 离心15分钟。微量移液器在不干扰沉淀的情况下尽可能多地去除上清液。 注意:颗粒会非常松散。</li>
		<li>向沉淀中加入 50 μL PBS 并重悬。再次以15,000-20,000× g 离心管15分钟。</li>
		<li>使用 10 μL 吸头，直接吸出 4 μL 沉淀，并将其转移到干净的 1.5 mL 管中（图 1D1）。每个网格使用 1 μL 洗涤和浓缩的金。 注意:使用的基准点类型取决于下游应用。对于 TEM 断层扫描，请使用 10 nm 金珠。对于软 X 射线断层扫描，请使用 200 nm 金珠。对于相关显微镜检查，请使用荧光乳胶珠。对于涉及冷冻FIB研磨的实验，基准点通常不是必需的，因为研磨过程会破坏它们。</li>
	</ol></li>
	<li>杂交<ol><li>将环境室设置为95%的湿度和30°C（图1D1）。使用 5/15 镊子选择网格。</li>
		<li>用PBS清洗网格并将它们转移到插入镊子上（图1D2）。固定后，取出 5/15 镊子并滑动夹子amp 在插入镊子上。</li>
		<li>将网格插入切入式冷冻机，保持夹子牢固（图 1D3）。向网格的背面加入1μL金基准点（图1D4）。将 2-3 μL PBS 添加到网格的碳侧。使用自动印迹法印迹网格的背面，并将冷冻液浸入液态乙烷中（图1D5）。 注:建议每个网格的体积在 3-4 μL 之间，以获得最佳印迹效果。洗涤后，网格可能会保留不同量的 PBS。调整增加的 PBS 体积，以考虑网格上预先存在的液体潴留。建议将黄金基准点添加到网格的背面。在前面加法会导致在插入点附近产生金基准点池，而从后面加法会导致更均匀的解决方案。印迹持续时间取决于网格的细胞牙科。空格格分别为 2 秒、正常格栅为 3 秒、全格格为 4 秒。可用的速冻仪类型可能决定了印迹的方法:徕卡GP2速冻仪是该协议中使用的速冻机，默认情况下能够进行自动单面印迹。Thermo Fisher Vitrobot 可进行自动双面印迹，但这通常会损坏附着在碳侧的细胞。也可以在 Vitrobot 中进行手动印迹，也可以使用手动重力柱塞。</li>
	</ol></li>
</ol>

用于原位细胞冷冻断层扫描的电子显微镜网格的制备

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作者声明没有利益冲突。

在这里，我们提供了一种可访问、灵活且可重复的方案，用于在原 位 冷冻电子断层扫描应用中的电子显微镜网格上接种细胞。该方法可以很容易地适应下游应用和/或实验要求的需求。除了极大的灵活性外，我们还描述了一种工作流程，该工作流程可以优化和减少网格接种中的常见缺陷，特别是对碳层的广泛破坏、低细胞密度和薄细胞突起的结构完整性差。
尽管此处描述的协议确实提供了几种替代方案，但应遵循一些关键步骤来优化一般结果。网格细胞接种的最大问题之一是网格从孔或微载玻片上分离和漂浮。因此，重要的是在两侧用粘附溶液完全润湿网格，并防止其在孵育期间干燥。如果使用 3D 打印的网格支架，请注意，对这些支架进行多次介质更换可能会产生浮动网格，因为被困在网格下方的空气会迫使其离开支架。 
我们选择的镊子还提高了网格质量，因为它提供了一种几何上有利的方法来操纵网格，而不会造成会损坏碳层的大量网格弯曲。在坠入之前，将细胞保持在37°C下尽可能长的时间可以减少细胞的痛苦，并改善网格上薄的可成像细胞的数量。最后，从金侧进行印迹将保护细胞免受可能导致脆弱细胞结构受损的苛刻机械力的影响。
虽然不包括在该协议中，但网格光微图案化已被证明通过优化可成像细胞与网格方块14中心的附着来增加可成像细胞的数量。最后，3D打印的网格支架最近被用于通过限制直接的网格操作来减少网格损坏12。
需要注意的是，该协议针对细胞的薄边缘和突起的成像进行了优化，用于冷冻断层扫描的应用。我们建议根据协议中的建议对各种情况进行故障排除，以便为所选的下游应用找到最佳结果。总体而言，该协议提供了一种可靠而通用的方法，可以将细胞接种到网格上，可以根据特定需求进行调整。

原位细胞冷冻断层扫描是一种强大的技术，无需化学固定即可研究细胞中的生物学相关结构。通过将细胞连接到EM网格并在制冷剂中对网格进行浸入冷冻，感兴趣的物体在其自然细胞环境中被冷冻，而不会从细胞内水中形成结晶冰1,2。化学固定和结晶冰的形成都会破坏相关分子的结构，例如蛋白质和脂质，从而降低使用这些技术获得的图像的生物学准确性3,4。在断层扫描中，使用电子显微镜以增量角度对网格进行成像，然后使用这些图像来构建成像的目标区域的三维表示5。原位冷冻断层扫描可与其他显微镜技术一起用于综合和相关成像，例如冷冻荧光成像、软 X 射线断层扫描和 cryoFIB/SEM（低温聚焦离子束/扫描电子显微镜）6,7,8,9,10,11 .与任何单一显微镜技术相比，集成多种技术可以获得更多关于结构或过程的信息。
尽管原 位 细胞冷冻断层扫描具有所有优点，但由于各种原因，样品制备可能具有挑战性。由于它们的脆弱性，强行操纵电子显微镜网格会导致损坏，特别是薄碳层很脆弱，容易撕裂，从而减小网格的可成像面积。由于尺寸小，电子显微镜网格也难以操作，并且容易与用于生长细胞的孔或微载玻片表面分离。由于井或微载玻片的几何形状，操作这些网格可能很困难。网格准备不当（例如，允许它们漂浮）会导致细胞密度低并减少潜在成像区域的数量，特别是当细胞不容易附着在网格本身时。对于直接细胞冷冻断层扫描，细胞必须扩散得非常薄，这可能会因多种原因而中断，包括温度不当或网格处理粗糙。
通过各种优化，本文介绍的技术旨在处理在制备冷冻断层扫描电子显微镜网格时出现的这些最常见的陷阱。使用 5/15 角镊子可以操纵孔板或微载玻片内的网格。在电镀前将纤连蛋白溶液涂在网格的两侧，可降低浮动网格的可能性，这有利于确保网格具有足够的细胞密度，并且网格不太可能因操作而损坏。通过将网格保持在 37°C 下孵育直到冷冻前，我们还确保细胞保持在舒适的环境中，以防止细胞缩回其薄边缘。从背面吸干网格还可以防止机械力对细胞的损坏。总之，这些措施提高了原 位 细胞冷冻断层扫描研究样品制备的成功率，增加了这种成像方法的可及性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 nm colloidal gold bead solution</td>
			<td>Sigma-Aldrich</td>
			<td>741957</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well multidish, 100/CS</td>
			<td>Fisher Scientific</td>
			<td>FB012927</td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra V-15R Benchtop Centrifuge, IVD 120 V 60 Hz</td>
			<td>Beckman-Coulter</td>
			<td>C63125</td>
			<td></td>
		</tr>
		<tr>
			<td>Au G300F1 with R2/2 Quantifoil carbon</td>
			<td>Quantifoil</td>
			<td>TEM-G300F1-AU</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>MilliporeSigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND&#160;counting chamber BLAUBRAND&#160;Neubauer improved</td>
			<td>Sigma-Aldrich</td>
			<td>BR717805-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>DMi1 Inverted Microscope</td>
			<td>Leica</td>
			<td>22A00G119</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle&#39;s medium - high glucose, no glutamine</td>
			<td>Gibco</td>
			<td>11-960-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont 5/15 tweezer</td>
			<td>Electron Microscopy Sciences</td>
			<td>0103-5/15-PO</td>
			<td></td>
		</tr>
		<tr>
			<td>EM GP2</td>
			<td>Leica</td>
			<td>587085</td>
			<td>Automated plunge freezer</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>A5209</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from bovine plasma, cell culture grade</td>
			<td>MilliporeSigma</td>
			<td>F1141</td>
			<td></td>
		</tr>
		<tr>
			<td>GenJet version II in vitro DNA transfection reagent</td>
			<td>SignaGen Laboratories</td>
			<td>SL100489</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX I 100x</td>
			<td>Fisher Scientific</td>
			<td>35050061</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Neslab EX-211 Heating Circulator</td>
			<td>Neslab</td>
			<td>Out of production</td>
			<td>Water bath for media warming</td>
		</tr>
		<tr>
			<td>Original Portable Pipet-Aid Pipette Controller</td>
			<td>Drummond Scientific</td>
			<td>4-000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pelco easyGlow device</td>
			<td>Pelco</td>
			<td>91000S</td>
			<td>Glow discharge device</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Pipetman P1000, 100&#8211;1000 &#181;L, Metal Ejector</td>
			<td>Gilson</td>
			<td>F144059M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P2, 0.2&#8211;2 &#181;L, Metal Ejector</td>
			<td>Gilson</td>
			<td>F144054M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman P20, 2&#8211;20 &#181;L, Metal Ejector</td>
			<td>Gilson</td>
			<td>F144056M</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman number 2 filter paper, 55 mm</td>
			<td>Whatman</td>
			<td>28455-041</td>
			<td>Blotting paper</td>
		</tr>
	</tbody>
</table>

sample preparation for in situ cryotomography of mammalian cells

原位 细胞冷冻断层扫描是一种强大的技术，用于在天然冷冻水合细胞环境中研究复杂物体，使其与细胞生物学和病毒学高度相关。将冷冻断层扫描与其他显微镜模式相结合的潜力使其成为综合和相关成像的完美技术。然而，原 位 细胞断层扫描的样品制备并不简单，因为细胞不容易附着在电子显微镜网格上并拉伸。此外，网格本身很脆弱，如果处理得太用力，可能会破裂，从而导致可成像区域的丢失。当用镊子操纵网格时，组织培养皿的几何形状也会带来挑战。在这里，我们描述了克服这些（和其他）挑战的技巧和窍门，并为贴壁哺乳动物细胞的 原位 细胞冷冻断层扫描和相关成像制备高质量的样品。随着冷冻显微镜技术的不断进步，这项技术在促进我们对复杂生物系统的理解方面具有巨大的前景。

在HIViGFPΔEnv和psPAX2共转染后，所有网格在碳层中的撕裂最小。在与转染试剂孵育 24 小时后，使用相光显微镜和荧光显微镜对网格进行成像（图 2）。模拟网格和共转染网格上的细胞在多个网格方块中都包含活细胞。
psPAX2 编码 HIV-1 的所有结构蛋白和酶蛋白，无需任何荧光标记。HIViGFPΔEnv 与 psPAX2 相似，但具有 GFP 标记的 HIV Gag 蛋白的代码。两种质粒都是ΔEnvelope。共转染导致荧光 HIV-1 颗粒的天然样组装和出芽，使其成为在生物安全 1 级条件下进行 HIV CLEM 研究的绝佳系统。共转染网格显示一部分细胞表现出绿色荧光，表明共转染成功。模拟网格上没有细胞表现出荧光，进一步验证了使用 HIViGFPΔEnv 和 psPAX2 的共转染。使用基于光学的显微镜观察网格后，将网格冷冻并移至液氮中长期储存。
图 3 描述了使用相同实验方法但使用略有不同的质粒构建体产生的网格的结果。使用不同的HIV克隆（HIVmCherryΔEnv和NL4-3ΔEnvGFP）以1:6的比例共转染含U2OS的网格。由于使用了更高质量量的荧光标记质粒，这些网格能够观察到更多的转染细胞，在使用 cryoCLEM 和 cryoET 捕获图像时具有优势。使用 cryoCLEM，使用冷冻荧光显微镜为每个网格生成完整的网格图谱，以记录所有共转染细胞的位置。在已知细胞位置的情况下，进行冷冻ET。收集完整的低倍率网格图谱，并与冷冻荧光时收集的荧光图谱叠加（图3A）。在细胞位点收集冷冻造影，捕捉病毒生命周期的复杂细节，包括HIV从细胞中组装和出芽（图3B）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65697/65697fig01.jpg"> 图 1:网格上的单元格播种工作流程。 在冷冻电镜网格上接种细胞的整个过程的示意图。该过程分为四个主要步骤，包括 （A） 在孔中制备用于接种的网格，（B） 向每个孔中添加适量的细胞，（C） 可选的细胞转染用于荧光成像，以及 （D） 网格的浸入冷冻以允许样品的玻璃化。 <a href="https://www.jove.com/files/ftp_upload/65697/65697fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65697/65697fig02.jpg"> 图 2:使用 HIViGFPΔEnv 和 psPAX2 共转染 U2OS。 U2OS 细胞与含 GFP 的 HIViGFPΔEnv 和 psPAX2 以 1:3 的比例共转染。通过相差和荧光显微镜对网格进行成像。显示具有GFP表达的细胞表明共转染成功。比例尺:500μm。比例尺（插图）:100 μm。 <a href="https://www.jove.com/files/ftp_upload/65697/65697fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65697/65697fig03.jpg"> 图 3:潜在的下游基于冷冻的方法 。 （A） 共转染 U2OS 细胞的 cryoCLEM 图像。绿色细胞代表产生HIV的细胞，用于测量共转染成功。红色点状物代表 mCherry 标记的 HIV-1 Gag。比例尺:250μm。比例尺（插图）:25 μm （B） 从 U2OS 细胞质膜萌芽的多个 HIV 颗粒的冷冻 ET 图像。比例尺:50纳米 <a href="https://www.jove.com/files/ftp_upload/65697/65697fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about Optimizing Grid Preparation for Enhanced Cryoelectron Tomography at JoVE.com

作者聚焦:优化增强型冷冻电子断层扫描的网格制备 

该方法为原 位 细胞冷冻断层扫描和相关光电子显微镜 （CLEM） 的电子显微镜 （EM） 网格的制备提供了一种可访问且灵活的协议。

哺乳动物细胞 原位 冷冻断层扫描的样品制备

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...

本研究的范围是开发一种既可访问又可靠可重复的样品制备方法。通过实现这一点，直接细胞冷冻电子断层扫描的力量可用于回答各种生物学问题。电网准备的挑战是广泛的。细胞不容易附着在网格上，操作会导致碳层损坏，从而导致可成像网格区域的丢失。此外，在不注意适当的样品制备条件的情况下，可以减少薄细胞突起的发生率。目前的协议通常需要专门的设备来解决接种的主要瓶颈，例如分别用于细胞附着和操作的微图案化和 3D 打印支架。该协议为解决方案提供了更大的可访问性，同时仍然保持了这些专用工具用于下游应用的灵活性。

sample-preparation-for-in-situ-cryotomography-of-mammalian-cells

Research

JoVE Journal

Biochemistry

Sample Preparation for In Situ Cryotomography of Mammalian Cells

2.8K 次观看.  University of Minnesota. 本研究的范围是开发一种既可访问又可靠可重复的样品制备方法。通过实现这一点，直接细胞冷冻电子断层扫描的力量可用于回答各种生物学问题。电网准备的挑战是广泛的。细胞不容易附着在网格上，操作会导致碳层损坏，从而导致可成像网格区域的丢失。此外，在不注意适当的样品制备条件的情况下，可以减少薄细胞突起的发生率。目前的协议通常需要专门的设备?...

视频: 作者聚焦:优化增强型冷冻电子断层扫描的网格制备 

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

基于板块的秀丽线虫规模化栽培: 糖尿病代谢变化研究的样品制备

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

An All-in-one Sample Holder for Macromolecular X-ray Crystallography with Minimal Background Scattering

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. A prerequisite for the method is that highly ordered crystalline specimen need to be grown from the macromolecule to be studied, which then need to be prepared for the diffraction experiment. This preparation procedure typically involves removal of the crystal from the solution, in which it was grown, soaking of the crystal in ligand solution or cryo-protectant solution and then immobilizing the crystal on a mount suitable for the experiment. A serious problem for this procedure is that macromolecular crystals are often mechanically unstable and rather fragile. Consequently, the handling of such fragile crystals can easily become a bottleneck in a structure determination attempt. Any mechanical force applied to such delicate crystals may disturb the regular packing of the molecules and may lead to a loss of diffraction power of the crystals. Here, we present a novel all-in-one sample holder, which has been developed in order to minimize the handling steps of crystals and hence to maximize the success rate of the structure determination experiment. The sample holder supports the setup of crystal drops by replacing the commonly used microscope cover slips. Further, it allows in-place crystal manipulation such as ligand soaking, cryo-protection and complex formation without any opening of the crystallization cavity and without crystal handling. Finally, the sample holder has been designed in order to enable the collection of in situ X-ray diffraction data at both, ambient and cryogenic temperature. By using this sample holder, the chances to damage the crystal on its way from crystallization to diffraction data collection are considerably reduced since direct crystal handling is no longer required.

A novel sample holder for macromolecular X-ray crystallography along with a suitable handling protocol is presented. The system allows crystal growth, crystal soaking and in situ diffraction data collection at both, ambient and cryogenic temperature without the need of any crystal manipulation or mounting.

用于大分子 X 射线晶体学的一体式样品支架,具有最小的背景散射

Macromolecular X-ray crystallography (MX) is the most prominent method to obtain high-resolution three-dimensional knowledge of biological macromolecules. ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

探头电喷电镀质谱的样品制备

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality have been seen for microcrystals with the development of beamline optics, beam stability and variable beam size focusing from submicron to microns, such as at the VMXm beamline at Diamond Light Source1. Further improvements in data quality will be gained through improvements in sample environment and sample preparation. Microcrystals inherently generate weaker diffraction, therefore improving the signal-to-noise is key to collecting quality X-ray diffraction data and will predominantly come from reductions in background noise. Major sources of X-ray background noise in a diffraction experiment are from their interaction with the air path before and after the sample, excess crystallization solution surrounding the sample, the presence of crystalline ice and scatter from any other beamline instrumentation or X-ray windows. The VMXm beamline comprises instrumentation and a sample preparation protocol to reduce all these sources of noise.
Firstly, an in-vacuum sample environment at VMXm removes the air path between X-ray source and sample. Next, sample preparation protocols for macromolecular crystallography at VMXm utilize a number of processes and tools adapted from cryoTEM. These include copper grids with holey carbon support films, automated blotting and plunge cooling robotics making use of liquid ethane. These tools enable the preparation of hundreds of microcrystals on a single cryoTEM grid with minimal surrounding liquid on a low-noise support. They also minimize the formation of crystalline ice from any remaining liquid surrounding the crystals. 
We present the process for preparing and assessing the quality of soluble protein microcrystals using visible light and scanning electron microscopy before mounting the samples on the VMXm beamline for X-ray diffraction experiments. We will also provide examples of good quality samples as well as those which require further optimization and strategies to do so.

The signal-to-noise ratio of data is one of the most important considerations in performing X-ray diffraction measurements from microcrystals. The VMXm beamline provides a low-noise environment and microbeam for such experiments. Here, we describe sample preparation methods for mounting and cooling microcrystals for VMXm and other microfocus macromolecular crystallography beamlines.

VMXm 光束线微晶样品制备管道

The mounting of microcrystals (&#60;10 &#181;m) for single crystal cryo-crystallography presents a non-trivial challenge. Improvements in data quality ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

密度梯度超速离心研究哺乳动物细胞内吞循环

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

冷冻电镜高温样品网格的制备

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

从哺乳动物细胞中纯化泛素化p53蛋白

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

用于冷冻电镜结构研究的链霉亲和素亲和网格的制备

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community.

作者聚焦:使用链霉亲和素-生物素方法增强冷冻电镜样品制备

A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron microscopy.

链霉亲和素亲和网格制备用于冷冻电子显微镜样品制备

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, ...

哺乳动物细胞 原位 冷冻断层扫描的样品制备

哺乳动物细胞原位冷冻断层扫描的样品制备