这项工作得到了德克萨斯州癌症研究预防研究所[RR150076]、国家科学基金会[1751174]的资助;国家卫生研究院 [NS-111236]， 韦尔奇基金会 [1-1923-20170325];卢佩·默奇森基金会、泰德·纳什长寿基金会和金氏基金会（西尔学者计划）

1. 哺乳动物细胞的培养和低温保护
<ol><li>培养和收获哺乳动物细胞<ol>
<li>解冻冷冻人类胚胎肾细胞（HEK 293）的一个无名小包。</li>
		<li>培养 HEK 293 细胞在生长介质 （例如， DMEM 与 10% 胎儿牛血清和 1% 笔链球菌） 在 37 °C 与 5% CO2 在 100 毫米板 2 至三段 （7-10 天）.</li>
		<li>将细胞和培养在150毫米的盘子中分裂，直到细胞达到90-95%的汇流。 注：150 mm 板在 > 90% 的汇流将足以填充两个直径为 3.2 mm 的蓝宝石转子。</li>
		<li>使用 4 mL 的试金石（见材料表）和 10 mL 的介质收集细胞。将悬架以 673 x g（1000 rpm）将悬架转移到无菌的 15 mL 圆锥形离心机中，在室温下 5 分钟。取出超高超。</li>
		<li>用磷酸盐缓冲盐水（PBS）（pH 7.4，+CaCl 2，+MgCl 2）清洗细胞颗粒。</li>
	</ol>
</li>
	<li>细胞的冷冻保护<ol>
<li>在微中心管中收集 50 μL 细胞颗粒。在单独的管子中准备 50 μL 的 PBS 和 18 μL 甘油的混合物。</li>
		<li>将甘油-PBS混合物加入颗粒顶部，轻轻敲击管侧，直到没有团块，将50微升细胞颗粒与68微升甘油-PBS混合物轻轻混合，并重新吸收颗粒。 注意：轻柔的管道也可以用来重新使用细胞，但是，确保细胞完整性不受影响。</li>
	</ol>
</li>
</ol>2. 在NMR转子中保存哺乳动物细胞
<ol><li>将细胞转移到 3.2 毫米蓝宝石转子中。<ol>
<li>要制作漏斗，切开一个 200 μL 移液器尖端，并将切口移液器尖端的窄端插入 3.2 mm 转子中。</li>
		<li>将细胞转移到位于转子上的漏斗中。将转子与漏斗一起放入微中心管中，在室温下以 673 x g 离心将细胞颗粒到转子底部 2-3 分钟。</li>
		<li>从转子中取出超高音和任何多余的样品。重复这两个步骤，直到转子完全充满细胞。 注：可选，在冻结前确定样品的细胞生存能力。在 100 μL 的无 FBS DMEM 中重复 10 μL 的多余细胞颗粒。将悬架的 10 μL 与 0.4% 试穿蓝色解决方案混合，并立即使用自动电池计数器评估可行性。仅使用 FBS 自由介质稀释细胞，因为血清会干扰尝试蓝色染色。</li>
		<li>使用市售包装工具用硅塞密封转子。</li>
		<li>通过垂直向下按压转子，用陶瓷驱动尖端关闭转子。避免触摸驱动器尖端侧面的细腻鳍。</li>
		<li>用银色永久标记标记蓝宝石转子底部边缘的一半，用黑色永久标记标记转子底部边缘的另一半，以便准确监控光谱仪内部转子的旋转。 注：转子标记不完善会妨碍对旋转频率的准确计数，导致无法实现稳定的旋转。因为标记不会写在冻结转子上，转子加热会损害样品的完整性，因此在冻结前标记转子是关键步骤。</li>
	</ol>
</li>
	<li>在 3.2 毫米蓝宝石转子内冷冻保存细胞。<ol>
<li>将一块纸巾或纸巾制成的垫子放在盖子下，放在低温小瓶的底部（见 材料表）。 注：纸巾保护转子标记免受低温小瓶两侧碰撞造成的损坏。</li>
		<li>将 3.2 毫米蓝宝石转子放入用纸巾填充的低温小瓶中，其显著端面向低温小瓶底部。</li>
		<li>将低温小瓶放入控制速率（-1 °C/min）冷却容器中，并将容器放入 -80 °C 的冰柜中，至少 3 小时，从而缓慢冻结转子。</li>
		<li>将含有冷冻转子的低温小瓶转移到液氮储存处。</li>
	</ol>
</li>
</ol>3. 冷冻样品低温转移到NMR光谱仪中
<ol><li>将冷冻样品运送到 NMR 设施。<ol>
<li>将含有冷冻转子的低温小瓶转移到充满液氮的小排华中，以便输送到 NMR 设施。</li>
	</ol></li>
	<li>将冷冻转子转移到液氮浴中。<ol>
<li>用 500 mL - 1 L 液氮填充干燥、隔热的宽嘴泡沫脱瓦尔。</li>
		<li>将转子从低温小瓶转移到充满液氮的宽口泡沫脱瓦尔中。<ol>
<li>将低温小瓶从手的转移去瓦尔，并举行它正上方的液氮，以保护它免受大气。</li>
			<li>一边拿着低温小瓶，嘴稍微向下指向，一边拧开盖子，让转子滑入液氮浴池。 注：一旦盖开，转子必须从低温小瓶迅速落入液氮浴（1秒以下），以防止冷凝物收集在转子上。液氮的蒸发在宽口泡沫中形成"氮云"，防止转子凝结，然后再浸入液氮中。转子对空气的长时间暴露会导致转子壁上的水分凝结，从而重新凝结成冰。</li>
		</ol>
</li>
	</ol></li>
	<li>转子低温转移至 NMR 样品捕手。<ol>
<li>将 1.5 mL 微中心管浸入液氮浴中，预切一个 1.5 mL 微中心管。不要关闭管子。 注：在转移到微中心管之前检查转子。使用钳子，将转子放在液氮表面正下方，检查转子标记是否完好无损，其墙壁上是否形成冰层，驱动尖端是否完好无损。转子壁上的冰晶沉积物显示为白色粉末。小心始终按其身体而不是驱动器尖端按住转子。不要用钳子尖划掉转子上的标记。</li>
		<li>在液氮浴的表面下，使用钳子将转子转移到微中心管中，驱动尖端朝向微中心管的底部，标记朝向管的开口。 注意：在浸入液氮或接触转子之前，请始终干燥钳子。</li>
		<li>使用钳子，将含有转子的管子按住颈部的液氮。</li>
		<li>手里拿着第二对钳子，准备将NMR样品捕手浸入液氮浴中，并抓住它，使其与微中心管的尖锐角度倾斜。 注意：尽量减少样品捕手与液氮接触的时间，以避免冻结 O 环。如果 O 环冻结，将很难将捕手插入光谱仪。</li>
	</ol></li>
	<li>低温转子从 NMR 样品捕获器传输到 NMR 光谱仪 注：这一步骤需要两个人，一个操作冷冻卡比尼，一个将样品从液氮转移到探针中。<ol>
<li>将冷冻卡比内特按在柜子上，使冷冻卡比内特处于弹出模式。 注：喷射模式清除从探针到大气的高压下干冷氮气流，以防止大气水分进入。</li>
		<li>将转子转移到 NMR 样品捕手。<ol>
<li>将 NMR 样品捕手的开端插入微中心管中，同时仍处于液氮表面之下。</li>
			<li>抬起微中心管和 NMR 样品捕手，让转子落入样品捕手。摇动 NMR 样品捕手和微中心管，以防转子卡在样品捕手的边缘。</li>
			<li>将空微中心管放在 NMR 样品捕手顶部，以保护转子不受空气影响。</li>
		</ol>
</li>
		<li>从探头中取出另一个空的 NMR 样品捕手，并将其放在地板上。</li>
		<li>将装有转子的 NMR 样品捕手转移到您的手上，取出微中心管并立即将其插入探针中。 注意：如果 O 环冻结，将很难收紧样品捕手。继续使用武力，直到它滑入原位。</li>
		<li>将操作冷冻卡比内特的人信号到"停止弹出"和"插入"。 注：插入模式将取样器中的转子引导到探针中。</li>
		<li>通过调整低温卡比内特控制的轴承和驱动流压力，将样品旋转到所需的旋转速率（例如 12 kHz）。（例如，立即将轴承气体增加到 ±200 mBar，将驱动气体增加到 10 mBar。样品旋转后，将轴承气体增加到 1000 mBar，将气体驱动到 200 mBar。旋转稳定时，将轴承增加到 2400 mBar，然后在几分钟内将驱动气体从 200 mBar 增加到 1700 mBar。VT 冷却气体恒定在 ±1070 L/h. 注：将微中心管和NMR样品捕手从液氮浴中取出时，请确保微中心管内有足够的液氮来包围转子。最大限度地缩短将转子转移到 NMR 样品捕手和将 NMR 样品捕手插入光谱仪之间的时间。3.4 中的所有步骤应在 30s 内完成。</li>
	</ol></li>
</ol>4. 从 NMR 光谱仪中低温去除样品
<ol><li>液氮浴和低温小瓶的准备<ol>
<li>将 500 mL - 1 L 液氮倒入宽口泡沫脱瓦尔，并将浴缸置于光谱仪下。</li>
		<li>预冷低温小瓶。将含有一块纸巾的空低温小瓶浸入液氮浴中。</li>
	</ol>
</li>
	<li>转子从探针到低温小瓶的低温转移<ol>
<li>通过降低驱动和轴承气体流，并通过切换到弹射模式弹出转子，将旋转速率降低到 0 kHz。</li>
		<li>保持弹出模式，从探针中取出样品捕手，将转子直接滴入含有液氮的宽口泡沫脱瓦尔中。</li>
		<li>使用预冷钳子，将转子转移到液氮表面下的预冷低温小瓶中。</li>
		<li>盖上低温小瓶。将低温小瓶盖浸入液氮中，以预切低温小瓶盖。从浴缸中取出含有转子和液氮的低温小瓶，用预切盖盖盖管。不要拧紧盖，以便可以安全地释放蒸发氮。</li>
		<li>将低温小瓶重新浸入液氮中。样品可以转移到较长期的液氮储存或立即拆开包装作进一步分析。</li>
	</ol>
</li>
</ol>5. 拆包转子和可行性测量
<ol><li>拆开转子和测量可行性<ol>
<li>预热血清免费介质 （DMEM） 或 PBS 至 37 °C。</li>
		<li>从液氮中取出转子。拆下驱动尖端和硅塞。 注：蓝宝石是一种优秀的热导体。避免用手指触摸转子，因为传热会导致局部冻结-解冻事件，损害细胞生存能力。</li>
	</ol>
</li>
	<li>测量可行性<ol>
<li>在转子中的冷冻细胞颗粒中加入 20 μL 的加热介质并重新悬回细胞。用移液器取下悬架，将悬架与 100 μL 介质混合。</li>
		<li>去除 10 μL 的电池悬架，并与等量的 0.4% 试用蓝色溶液（v/v）混合。在室温下孵育30秒至1分钟。</li>
		<li>使用自动电池计数器测量可行性。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Costello, W. N., Xiao, Y., Frederick, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNP-Assisted+NMR+Investigation+of+Proteins+at+Endogenous+Levels+in+Cellular+Milieu.">DNP-Assisted NMR Investigation of Proteins at Endogenous Levels in Cellular Milieu.</a> Methods Enzymology. 615 (1), 373-406 (2019).</li><li>Hall, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization-Enhanced+NMR+Spectroscopy+of+Biomolecules+in+Frozen+Solution.">Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution.</a> Science. 276 (5314), 930-932 (1997).</li><li>Akbey, &. #. 2. 2. 0. ;., Oschkinat, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+applications+of+solid+state+MAS+DNP+NMR.">Structural biology applications of solid state MAS DNP NMR.</a> Journal of Magnetic Resonance. 269 (1), 213-224 (2016).</li><li>Matsuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helium-cooling+and+-spinning+dynamic+nuclear+polarization+for+sensitivity-enhanced+solid-state+NMR+at+14T+and+30K.">Helium-cooling and -spinning dynamic nuclear polarization for sensitivity-enhanced solid-state NMR at 14T and 30K.</a> Journal of Magnetic Resonance. 225 (1), 1-9 (2012).</li><li>Miyamoto, Y., Ikeuchi, M., Noguchi, H., Hayashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+Cryopreservation+of+Human+and+other+Mammalian+Cells+at+-80+&#176;C+for+8+Years.">Long-term Cryopreservation of Human and other Mammalian Cells at -80 &#176;C for 8 Years.</a> Cell Medicine. 10 (1), 1-7 (2018).</li><li>Lopez del Amo, J. M., Schneider, D., Loquet, A., Lange, A., Reif, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryogenic+solid+state+NMR+studies+of+fibrils+of+the+Alzheimer's+disease+amyloid-&#946;+peptide:+perspectives+for+DNP.">Cryogenic solid state NMR studies of fibrils of the Alzheimer's disease amyloid-&#946; peptide: perspectives for DNP.</a> Journal of Biomolecular NMR. 56 (4), 359-363 (2013).</li><li>Gupta, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+nuclear+polarization+enhanced+MAS+NMR+spectroscopy+for+structural+analysis+of+HIV-1+protein+assemblies.">Dynamic nuclear polarization enhanced MAS NMR spectroscopy for structural analysis of HIV-1 protein assemblies.</a> The Journal of Physical Chemistry B. 120 (2), 329-339 (2016).</li><li>Lim, B. J., Ackermann, B. E., Debelouchina, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targetable+Tetrazine-Based+Dynamic+Nuclear+Polarization+Agents+for+Biological+Systems.">Targetable Tetrazine-Based Dynamic Nuclear Polarization Agents for Biological Systems.</a> ChemBioChem. 21 (9), 1315-1319 (2020).</li><li>Jaudzems, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+nuclear+polarization-enhanced+biomolecular+NMR+spectroscopy+at+high+magnetic+field+with+fast+magic-angle+spinning.">Dynamic nuclear polarization-enhanced biomolecular NMR spectroscopy at high magnetic field with fast magic-angle spinning.</a> Angewandte Chemie International Edition. 57 (25), 7458-7462 (2018).</li><li>Chaytor, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibiting+ice+recrystallization+and+optimization+of+cell+viability+after+cryopreservation.">Inhibiting ice recrystallization and optimization of cell viability after cryopreservation.</a> Glycobiology. 22 (1), 123-133 (2012).</li><li>Mazur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+water+loss+from+cells+at+subzero+temperatures+and+the+likelihood+of+intracellular+freezing.">Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing.</a> The Journal of General Physiology. 47 (2), 347-369 (1963).</li><li>Lovelock, J. E., Bishop, M. W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Freezing+Damage+to+Living+Cells+by+Dimethyl+Sulphoxide.">Prevention of Freezing Damage to Living Cells by Dimethyl Sulphoxide.</a> Nature. 183 (4672), 1394-1395 (1959).</li><li>Bald, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relative+merits+of+various+cooling+methods.">The relative merits of various cooling methods.</a> Journal of Microscopy. 140 (1), 17-40 (1985).</li><li>Akiyama, Y., Shinose, M., Watanabe, H., Yamada, S., Kanda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryoprotectant-free+cryopreservation+of+mammalian+cells+by+superflash+freezing.">Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing.</a> Proceedings of the National Academy of Sciences. 116 (16), 7738-7743 (2019).</li><li>Shi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+non-contact+vitrification+of+cell-laden+droplets+based+on+cell+printing.">High-throughput non-contact vitrification of cell-laden droplets based on cell printing.</a> Scientific Reports. 5 (1), 17928 (2015).</li><li>Pegg, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+cryopreservation.">Principles of cryopreservation.</a> Methods in Molecular Biology. 1257, 3-19 (2015).</li><li>Ramilo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+the+covalent+intermediate+of+UDP-N-acetylglucosamine+enolpyruvyl+transferase+by+solution-state+and+time-resolved+solid-state+NMR+spectroscopy.">Detection of the covalent intermediate of UDP-N-acetylglucosamine enolpyruvyl transferase by solution-state and time-resolved solid-state NMR spectroscopy.</a> Biochemistry. 33 (50), 15071-15079 (1994).</li><li>Jeon, J., Thurber, K. R., Ghirlando, R., Yau, W. M., Tycko, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+millisecond+time-resolved+solid+state+NMR+to+the+kinetics+and+mechanism+of+melittin+self-assembly.">Application of millisecond time-resolved solid state NMR to the kinetics and mechanism of melittin self-assembly.</a> Proceedings of the National Academy of Sciences. 116 (34), 16717 (2019).</li><li>Pievo, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+freeze-quench+setup+for+multi-frequency+EPR+spectroscopy+of+enzymatic+reactions.">A rapid freeze-quench setup for multi-frequency EPR spectroscopy of enzymatic reactions.</a> Chemphyschem. 14 (18), 4094-4101 (2013).</li><li>Cherepanov, A. V., de Vries, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsecond+freeze-hyperquenching:+development+of+a+new+ultrafast+micro-mixing+and+sampling+technology+and+application+to+enzyme+catalysis.">Microsecond freeze-hyperquenching: development of a new ultrafast micro-mixing and sampling technology and application to enzyme catalysis.</a> Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1656 (1), 1-31 (2004).</li><li>Hu, K. N., Tycko, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+can+solid+state+NMR+contribute+to+our+understanding+of+protein+folding.">What can solid state NMR contribute to our understanding of protein folding.</a> Biophysical Chemistry. 151 (1), 10-21 (2010).</li><li>Hu, K. N., Yau, W. M., Tycko, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+a+transient+intermediate+in+a+rapid+protein+folding+process+by+solid-state+nuclear+magnetic+resonance.">Detection of a transient intermediate in a rapid protein folding process by solid-state nuclear magnetic resonance.</a> Journal of the American Chemical Society. 132 (1), 24-25 (2010).</li><li>Johnson, S., Nguyen, V., Coder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cell+viability.">Assessment of cell viability.</a> Current Protocols in Cytometry. 64 (1), 1-26 (2013).</li><li>Frederick, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity-enhanced+NMR+reveals+alterations+in+protein+structure+by+cellular+milieus.">Sensitivity-enhanced NMR reveals alterations in protein structure by cellular milieus.</a> Cell. 163 (3), 620-628 (2015).</li><li>Van Der Cruijsen, E. A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+DNP-supported+NMR+spectroscopy+using+Site-directed+spin+labeling.">Biomolecular DNP-supported NMR spectroscopy using Site-directed spin labeling.</a> Chemistry. 21 (37), 12971-12977 (2015).</li><li>Schlagnitweit, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+an+antisense+drug+complex+in+intact+human+cells+by+in-cell+NMR+spectroscopy.">Observing an antisense drug complex in intact human cells by in-cell NMR spectroscopy.</a> ChemBioChem. 20 (19), 2474-2478 (2019).</li><li>Debelouchina, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+nuclear+polarization-enhanced+solid-state+NMR+spectroscopy+of+GNNQQNY+nanocrystals+and+amyloid+fibrils.">Dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of GNNQQNY nanocrystals and amyloid fibrils.</a> Physical Chemistry Chemical Physics : PCCP. 12 (22), 5911-5919 (2010).</li><li>Schmidt, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submillisecond+freezing+permits+cryoprotectant-free+EPR+doubled+electron-electron+resonance+spectroscopy.">Submillisecond freezing permits cryoprotectant-free EPR doubled electron-electron resonance spectroscopy.</a> ChemPhysChem. 21, 1224-1229 (2020).</li></ol>

作者没有什么可透露的。

冷冻样品低温转移到NMR光谱仪中，通过NMR数据采集成功地保持了冷冻哺乳动物细胞的生存能力。该方法的成功表现在 MAS NMR 生存能力测量的前期和后期。这种方法是成功的，可概括到任何温度波动可能损害样品完整性的系统。当前提交的协议是与 HEK 293 电池线一起执行的。由于许多哺乳动物细胞系的低温保存条件非常相似，因此这里报告的条件很可能可以翻译成其他细胞系统：然而，它们可能需要进一步优化低温保护剂、样本量和冷冻率才能达到相同的结果。
这种方法可以通过在NMR实验后更快地拆开转子来改进。此步骤目前处于次优状态，其执行会影响细胞的生存能力。在将电池重新悬用于介质之前，必须从转子中取出驱动尖端和硅塞。当转子在拆卸驱动尖端时保持不均匀解冻，硅塞因此缩短转子处理时间会导致更高的振动。转子支架或其他工具的开发，以促进均匀解冻和快速去除驱动尖端和硅塞将有助于使NMR后可行性评估更准确。
本文中描述的低温样品加载方法仅限于支持在 NMR 磁体孔中放置探头的样品插入和弹出的 NMR 探头。虽然外部样品插入和弹出是商业 DNP 系统的标准配置，但自定义探头并不总是有此选项。此外，这种低温样品加载方法可能需要对构建与不同尺寸转子兼容的 NMR 探头进行一些修改。此协议已优化为 3.2 mm 转子，如果样品捕手的外径超过微中心管的内径（例如第 3.4.2 步），则可能需要修改。
随着DNP在MAS NMR中的应用，现在有可能在内源生理浓度24、25、26中检测蛋白质和其他生物分子。这为研究其原生环境中的生物分子开辟了可能性。在整个实验过程中保持细胞完整性和生存能力对于将光谱学的实验结果与生物现象联系起来可能至关重要。含有纯化蛋白或细胞解剖剂的样品不受控制冻结通常不会损害样品质量7，27，虽然有迹象表明，即使在纯化系统28中，冷冻率也可能是一个重要变量。然而，如果保持细胞完整性和生存能力对于解释很重要，哺乳动物细胞的样本需要以受控的速度冷冻。在这里，我们提出了一个协议，冻结和转移哺乳动物细胞的冷冻样本到预冷却的DNP MAS NMR仪器，以避免潜在的破坏性温度波动，并支持对可行的细胞的测量。

引入动态核极化用于神奇角度旋转核磁共振光谱，可将MAS NMR的灵敏度提高几个数量级。这使得生物分子在生理浓度或接近其生理浓度时能够检测。DNP可以而且确实提供在复杂生物环境中内源性（+1 μM）浓度下检测同位素标记蛋白所需的灵敏度。由于有成熟的协议，将同位素标记分子引入未标记的哺乳动物细胞，而不影响其生存能力，这为研究同位素丰富的生物分子在其原生环境中的内生水平开辟了可能性。此外，由于DNP增强在2、3、4的低温下效率更高，DNP MAS NMR的实验温度与长期储存可存活哺乳动物细胞5所需的温度完全一致。然而，将样品转移到DNP MAS NMR光谱仪的传统方法使其受制于破坏哺乳动物细胞的温度波动率。
MAS NMR实验要求样品以等于或大于同向相互作用的幅度的频率旋转，使其平均为零，通常至少为4千赫，通常高得多的6，7，8，9。因此，样品被包装成转子，其中有一个鳍尖，用于通过气体流驱动转子的旋转，并在另一端有一个标记，以便旋转频率可以通过转速计进行监控。大多数 MAS NMR 仪器的样品传输是通过将仪器外部的转子注入 NMR 探头末端的支架，并注入干燥空气或氮气流来完成的。转子到达以神奇角度容纳转子的天体后，样品旋转由空气涡轮机制推动。单独的气体支持流，推动和控制转子的温度。将转子插入 NMR 光谱仪并实现稳定的 MAS 旋转需要精细加工的驱动尖端和严格控制单独气体流的温度和压力。尽管有这些技术要求，但用于室温应用的商业 MAS NMR 探头的插入和实现稳定的 MAS 基本上实现了自动化。
然而，对于低温应用来说，情况更为复杂。用于低温应用的样品通常在室温下插入光谱仪，并冻结在天线器中。在第一分钟，样品温度迅速下降（> ±100 °C/分钟），系统温度需要几分钟才能平衡。由于温度和压力的相互作用，所需 MAS 的插入和接近通常用于低温应用手动处理。尽管需要人工干预，但冻结仪器内部的转子是有益的，因为它最大限度地减少了水和冷凝到探针中，这对成功旋转至关重要。环境湿气产生的凝结和结冰不仅能阻断转子本身的气体线、凝结或霜冻，还能机械地防止 MAS。因此，低温 MAS NMR 样品通常以超过 -100 °C/分钟的速率冻结在仪器内部。
如果冷却速度慢于5、10、11、12，则哺乳动物细胞可以通过冻结-解冻周期保持完整性，速率等于或慢于 1°C/分钟。或者，如果冷却速率超快13、14、15，则电池也会保持其完整性，冷却速率高于 104 °C/分钟。率中间到这两个极端破裂和杀死哺乳动物细胞由于冰晶形成内外的细胞，即使在低温保护剂16的存在。预冷却探针内室温转子的样品冷却速率在这两个极端之间下降，因此，为了研究低温保存完好的哺乳动物细胞，样品必须在转移到仪器之前冷冻并转移到仪器中，而不会出现温度波动，从而损害转子上的样品或霜冻积累，从而阻止转子旋转。该协议描述了一种无霜、预冷转子插入低温MAS NMR系统的方法，用于研究低温保存完好的存活哺乳动物细胞样本。此处描述的低温样本转移是为活体完整细胞的 NMR 特征而开发的。但是，它适用于温度波动可能损害样品完整性的任何系统。这包括任何复杂的系统，如冷冻淬火反应的化学和结构特征的被困反应中间体17，18，酶19，20或蛋白质折叠21，22。

<table>
	<tbody>
		<tr>
			<td>0.4% Trypan blue stain</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm cell culture dish</td>
			<td>Thomas Scientific</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm cell culture dish</td>
			<td>Nunc</td>
			<td>157150</td>
			<td></td>
		</tr>
		<tr>
			<td>3.2 mm sapphire rotor</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>45 &#176; angled forceps</td>
			<td>Hampton Research</td>
			<td>HR4-859</td>
			<td></td>
		</tr>
		<tr>
			<td>AMUPol</td>
			<td>Cortecnet</td>
			<td>C010P005</td>
			<td></td>
		</tr>
		<tr>
			<td>Black and Silver marker</td>
			<td>Sharpie</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cap removing tool</td>
			<td>Bruker ?</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>HyClone</td>
			<td>SH30529.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic cap</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CoolCell</td>
			<td>Corning/ Biocision</td>
			<td>UX-04392-00 (Corning) / BCS-405 (Bioscion)</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10288</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess II automated cell counter</td>
			<td>Invitrogen</td>
			<td>AMQAF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogen tubes</td>
			<td>Nalgene</td>
			<td>03-337-7Y</td>
			<td></td>
		</tr>
		<tr>
			<td>d8-glycerol</td>
			<td>Aldrich</td>
			<td>447498</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide, 99.8 % atom D</td>
			<td>Aldrich</td>
			<td>756822-1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>DNP NMR system with 1.7 T cryogen free gyrotron</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foam dewar</td>
			<td>Spearlab</td>
			<td>FD-800</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimwipes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Packaging tool</td>
			<td>Bruker ?</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Powdered PBS</td>
			<td>VWR</td>
			<td>VWRRV0780</td>
			<td></td>
		</tr>
		<tr>
			<td>Protonated PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon plug</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Vessel forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tryp-L</td>
			<td>Gibco</td>
			<td>12605010</td>
			<td></td>
		</tr>
	</tbody>
</table>

cryogenic sample loading into a magic angle spinning nuclear magnetic resonance spectrometer that preserves cellular viability

动态核极化 （DNP） 可显著提高神奇角度旋转 （MAS） 核磁共振 （NMR） 光谱的灵敏度。这些灵敏度随着温度的降低而增加，并且足够大，能够在大多数商用 DNP 配备的 NMR 光谱仪的运行温度 （+100 K） 下对浓度非常低的分子进行研究。这导致细胞内结构生物学的可能性，冷冻保存的细胞大分子在其内源水平在其本地环境中。但是，在 DNP MAS NMR 的典型样品处理过程中，细胞低温保存所需的冻结率超过，这导致细胞完整性和生存能力的丧失。本文描述了将哺乳动物细胞冷冻样本制备和低温转移到MAS NMR光谱仪的详细方案。

将哺乳动物细胞的冷冻前样本低温插入NMR光谱仪，支持整个NMR实验的可行性。 图1显示冷冻样品低温转移到预冷却的NMR探头的原理图。细胞的生存能力和完整性可以使用各种方法进行评估。在这里，我们使用了标准染料为基础的膜完整性测量，这与其他方法23非常一致。精密细胞不透水，可尝试蓝色，而膜完整性受损的细胞则可渗透。可使用自动电池计数器快速评估可尝试蓝渗透和不透水细胞的数量。使用此处描述的协议，MAS NMR（即点 5.2.3）之后哺乳动物细胞的锥蓝渗透性类似于哺乳动物细胞在任何温度变化之前的尝试蓝渗透性（即点 2.1.3）。但是，如果细胞被缓慢冻结，然后在插入前加热到室温（即在将转子加热到室温之前，再将转子加热到室温，然后插入冰冷探头），则通过 trypan Blue 评估的细胞生存能力将降低到细胞的 10% 以下（图 2）。因此，光谱仪内的冷冻细胞会导致细胞膜完整性丧失，而哺乳动物细胞冷冻样本的低温插入则支持细胞在整个NMR实验中的生存能力。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61733/61733fig01.jpg"> 图1：冷冻样品低温转移成预冷却NMR探针的示意图。（A） 接近液氮表面，拧开低温小瓶的顶部。（B） 将转子滑入液氮浴中。（C） 使用钳子将微中心管浸泡在原位，直到完全冷却。（D） 将转子插入微中心，驱动尖端朝向管底。用钳子推，而不是抓住。（E） 将微中心管握在液氮浴表面正下方，直观地检查转子，以确保转子无霜且标记良好。（F） 以高于表面的角度将样品捕手抱住。（G） 一旦样品捕手在微中心管内，立即将样品捕手和管子从液氮中取出。（H） 如果转子被夹在边缘上，摇动样品捕手。（I） 从探头中取出空样品捕手，放在地上。（J） 将微中心管从样品捕手中取出，内侧转子，插入、拧紧探针中的样品捕手，并在控制控制台上按"插入"。 <a href="https://www.jove.com/files/ftp_upload/61733/61733fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61733/61733fig02.jpg"> 图2：对哺乳动物细胞预冷冻样本的低温插入，可以测量细胞完整性，类似于从未冷冻过的哺乳动物细胞样本。从未冷冻过的样品（例如第 2.1.3 步）的试穿蓝色不透水细胞的百分比与 MAS NMR 之后的样本细胞（例如第 5.2.3 步，12 kHz MAS）相似。在插入预先冷却到 100 K 的 NMR 仪器之前，加热到室温的慢速冷冻细胞（例如第 3 步）在 MAS NMR 与 12 kHz MAS 之后的完整细胞百分比要低得多。 <a href="https://www.jove.com/files/ftp_upload/61733/61733fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability at JoVE.com

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

这里介绍了冷冻样品低温传输到动态核极化 （DNP） 神奇角度旋转 （MAS） 核磁共振 （NMR） 探测器的协议。该协议包括实验前转子存储方向和实验前后可行性测量方向。

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

cryogenic-sample-loading-into-magic-angle-spinning-nuclear-magnetic

Research

JoVE Journal

Biology

3.3K 次观看.  University of Texas Southwestern Medical Center. 这里介绍了冷冻样品低温传输到动态核极化 （DNP） 神奇角度旋转 （MAS） 核磁共振 （NMR） 探测器的协议。该协议包括实验前转子存储方向和实验前后可行性测量方向。

视频: Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

小鼠卵母细胞的细胞核移植到

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

科研

生物学

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

大会，加载和分析超速离心机样品池对齐

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

由不稳定的多蛋白质复合物的分离在体内控制细胞的交联和免疫磁性亲和层析

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

代谢物的浓度从低密度浮游社区的环境代谢，利用核磁共振光谱

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Quantifying Mixing using Magnetic Resonance Imaging

Mixing is a unit operation that combines two or more components into a homogeneous mixture. This work involves mixing two viscous liquid streams using an in-line static mixer. The mixer is a split-and-recombine design that employs shear and extensional flow to increase the interfacial contact between the components. A prototype split-and-recombine (SAR) mixer was constructed by aligning a series of thin laser-cut Poly (methyl methacrylate) (PMMA) plates held in place in a PVC pipe. Mixing in this device is illustrated in the photograph in Fig. 1. Red dye was added to a portion of the test fluid and used as the minor component being mixed into the major (undyed) component. At the inlet of the mixer, the injected layer of tracer fluid is split into two layers as it flows through the mixing section. On each subsequent mixing section, the number of horizontal layers is duplicated. Ultimately, the single stream of dye is uniformly dispersed throughout the cross section of the device.
 Using a non-Newtonian test fluid of 0.2% Carbopol and a doped tracer fluid of similar composition, mixing in the unit is visualized using magnetic resonance imaging (MRI). MRI is a very powerful experimental probe of molecular chemical and physical environment as well as sample structure on the length scales from microns to centimeters. This sensitivity has resulted in broad application of these techniques to characterize physical, chemical and/or biological properties of materials ranging from humans to foods to porous media 1, 2. The equipment and conditions used here are suitable for imaging liquids containing substantial amounts of NMR mobile 1H such as ordinary water and organic liquids including oils. Traditionally MRI has utilized super conducting magnets which are not suitable for industrial environments and not portable within a laboratory (Fig. 2). Recent advances in magnet technology have permitted the construction of large volume industrially compatible magnets suitable for imaging process flows. Here, MRI provides spatially resolved component concentrations at different axial locations during the mixing process. This work documents real-time mixing of highly viscous fluids via distributive mixing with an application to personal care products.

Magnetic resonance imaging (MRI) provides a powerful tool to evaluate the effectiveness of process equipment during operation. We discuss the use of MRI to visualize mixing in a static mixer. The application is relevant to personal care products, but can be applied to a broad range of food, chemical, biomass and biological fluids.

量化混合使用磁共振成像

Mixing is a unit operation that combines two or more components into a homogeneous mixture.  This work involves mixing two viscous liquid streams using an ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

细胞形态变化，从三维荧光图像量化的分析工具，

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

透射电镜的小型化样品制备方法

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...