这项工作得到了通过DOM程序和格兰特211684（BACSIN）来自欧盟社区FP7框架计划战略环境研究（MISTRA）的瑞典基金会。我们感谢J. Engstrand的协助拍摄的X射线分析和研究唐与概念的叙述帮助。

1。种植和收获的P恶臭 <ol><li>准备起动恶臭假单胞菌文化与体育的殖民地接种100毫升的胰蛋白酶大豆肉汤（TSB） 恶臭细胞在琼脂上培养，用胰蛋白酶大豆肉汤（TSA）的补充。保持在30°C的文化在摇板设置在130转。 </li><li> 7小时后从起始培养相当于主培养的最终体积为1/10的等分试样转移到新鲜TSB培养基。在30℃下在摇板在每分钟130转的保持细胞16小时。 </li><li>生长16小时后收获细胞通过细胞培养物以1500×g，20分钟，在RT纺丝。倒出上清液，并洗涤细胞重新悬浮细胞沉淀中的NaCl溶液是等渗的生长介质。在这种情况下，使用150 mM氯化钠溶液。然后将细胞离心一次，并倾出上层液重悬细胞之前，在制定介质见第3.2步。 </li></ol> 2。其他种类的培养<ol><li>为了适应协议其他细菌​​物种的种植和收获过程应根据该品种的要求改变。为了避免渗透休克处理细胞时，在收获和洗涤步骤（次），渗透压的解决方案需要相匹配的生长培养基中收获。 </li></ol> 3。配方细胞<ol><li>准备制定解决方案权衡各自的基质成分，溶解在水中。为了达到等渗条件下，调整量的赋形剂，或者添加NaCl或其他一些细胞的相容性溶质，以达到所需的重量克分子渗透压浓度。二糖等低分子量物质收入的金额能够很容易地进行调整，以达到等渗条件。对于高分子量的聚合物的物质，如单元格兼容，因此必须进行调整，等渗琵琶， 如 NaCl或双糖。需要注意的是任何一种物质除了会影响制剂的物理性质，这反过来又可能影响冷冻干燥行为和细胞生存。 </li><li>倒出洗涤液（在这种情况下，氯化钠），并在各自的制剂媒体重新悬浮细胞。 </li><li>确保细胞均匀地分散。低粘度的制剂的涡旋，而粘度较高的制剂中添加， 例如 ，搅拌棒和摇动容器，直到达到均匀混合，混合使用。 </li><li>把配方转化为冻干机小瓶。小瓶应称量空的，用样品之前和之后冷冻干燥，冷冻干燥过程中除去的水的量计算。 </li><li>如果被密封小瓶冷冻机内添加橡胶塞的小瓶，请参见第4.3步。 </li><li>枚举冷冻干燥前的各配方中的单元格，请参阅步骤6。 </li> &lt;/ OL&gt; 4。冷冻干燥<ol><li>的冷冻干燥条件下进行调整，以制剂的物理性质。最重要的参数是在这种情况下，玻璃化转变温度Tg，该制剂，记的Tg&#39;为冷冻浓缩的样品表明，水是仍然存在。容易使用差示扫描量热法（DSC）测定的冻干浓缩制剂的Tg&#39;，请参阅第7步。 </li><li>调整冷冻干燥过程的参数，以使样品的温度总是低于样品的Tg，腔室压力允许快速的冰升华，即初级干燥，在制剂中的残留水， 即二次干燥。如果样品温度Tg以上，在干燥过程中，有很大的样品结构崩溃的风险，这可能显着减少细胞存活。 </li><li> F之后，保护干产品reeze干燥样品气氛已被控制。如果可能的话，密封样品在冷冻机的冷冻干燥周期结束之前，释放真空。或者可以填充小瓶，用一个优选的气氛。这是很重要的，以避免暴露的样品，因为任何湿气或存储的气氛中存在的氧的环境条件下会影响细胞的生存。 </li><li>称重的小瓶。 </li></ol> 5。补液<ol><li>用无菌去离子水再水合样品。要添加的水的量应该是冷冻干燥过程中除去空的小瓶中，冷冻干燥之前和之后的相同的小瓶样品的重量计算出的量相同。涡样品，直到解决方案，然后再出现同质。 </li></ol> 6。列举<ol><li>绘制从每个样品中加入100μl，并进行10倍系列稀释。从集成的稀释其余100微升镀在TSA板。将板温育所需的温度和时间。 </li></ol> 7。制定冻结行为表征<ol><li>取少量5 - 10毫克的样品，并在DSC-铝盖锅附上。准备一个空锅作为参考。 </li><li>将DSC温度扫描程序。冷却的程序被设置为模仿的冷冻步骤中的冷冻干燥程序。这是通过作为冷却动力学影响的水合制剂的Tg&#39;。加热前扫描开始打倒的温度远低于预期的Tg&#39;被调查的配方，通常为-100°C。 </li><li>选择适当的加热速度，通常是升温速率为5 - 40℃/ min的使用。以瓦为单位的记录，可热流信号，并会受加热速率。较高的加热速率将给予较大的信号TG&#39;。 </li><li>设定的温度范围，这样我吨涵盖TG的&#39;的制定和熔化。 </li></ol> 8。水合配方的表面张力测量<ol><li>本实验中使用的实验装置是根据杜诺氏方法测量表面。 </li><li>清洁铂金戒指和仔细测量容器。该船是用丙酮冲洗，擦拭无绒布组织，用无色的火焰，然后在里面燃烧。在无色火焰环呈红色。 </li><li>填写船只与解决方案，并让表面测量前定居。很长一段时间后，才可达到平衡状态的解决方案， 如聚合物，用在这里简单的设置将提供定性数据。 </li><li> HEC或HPMC的配方具有高的粘度， 例如，解决方案中的2％（w / w的），表面张力，必须测量在较低的浓度的溶液，然后外推。这是可以做到的事实，通过利用的浓度低于0.5％（重量/重量）的表面张力水平的下降。 </li></ol> 9。 X射线分析的干制剂<ol><li>取少量矩阵/细菌，并将其加载到样品架。 </li><li>安装到的X-射线衍射仪样品架。 </li><li>结晶相存在于样品中分析任何来自于Bruker EVA的程序包都可以使用。 </li></ol> 10。电子显微镜<ol><li>取少量矩阵/细菌的小瓶，并修复它的SEM存根上。 </li><li>加载SEM持有人在溅射涂布机（Thermo VGScientific的极化子SC7640，在本工作中使用的）和涂布样品用几nm的Au / Pd或Pt。在这些实验中的溅射参数为：​​1,900 V，20毫安和20秒，约5 - 10 nm的金/钯涂层。涂层减少电动充电过程中的图像采集。建议开始薄的涂层，如果的充电仍然存在，并导致图像工件，样品应再次被涂覆。 </li><li>加载SEM持有人在SEM室。选择合适的成像参数。对于图2中所用的仪器（FEI地层DB235）中，使用5 kV的加速电压，spotsize 3，工作距离为5毫米，二次电子探测器。 </li><li>可选地，这是可能的，使用聚焦离子束切割嵌入聚合物中的细菌，进一步揭示了细节的横截面。 </li></ol>

<ol>
	<li>Wessman, P., Mahlin, D., 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=Impact+of+matrix+properties+on+the+survival+of+freeze-dried+bacteria.">Impact of matrix properties on the survival of freeze-dried bacteria.</a> J. Sci. Food Agric. 91 (14), 2518-2528 (2011).</li><li>Ablett, S., Izzard, M. 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=Differential+Scanning+Calorimetric+Study+of+Frozen+Sucrose+and+Glycerol+Solutions.">Differential Scanning Calorimetric Study of Frozen Sucrose and Glycerol Solutions.</a> Journal of the Chemical Society-Faraday Transactions. 88 (6), 789-794 (1992).</li><li>Knopp, S. A., Chongprasert, S., 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=The+relationship+between+type+TMDSC+curve+of+frozen+sucrose+solutions+and+collapse+during+freeze-drying.">The relationship between type TMDSC curve of frozen sucrose solutions and collapse during freeze-drying.</a> Journal of Thermal Analysis and Calorimetry. 54 (2), 659-672 (1998).</li><li>Crowe, J. H., Leslie, S. B., 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=Is+Vitrification+Sufficient+to+Preserve+Liposomes+during+Freeze-Drying.">Is Vitrification Sufficient to Preserve Liposomes during Freeze-Drying.</a> Cryobiology. 31 (4), 355-366 (1994).</li><li>Jain, P., Sen, S., 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=Effect+of+glass-forming+biopreservatives+on+head+group+rotational+dynamics+in+freeze-dried+phospholipid+bilayers:+A+P-31+NMR+study.">Effect of glass-forming biopreservatives on head group rotational dynamics in freeze-dried phospholipid bilayers: A P-31 NMR study.</a> Journal of Chemical Physics. 131 (2), (2009).</li><li>Stock, J. B., Rauch, B., 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=Periplasmic+Space+in+Salmonella-Typhimurium+and+Escherichia-Coli.">Periplasmic Space in Salmonella-Typhimurium and Escherichia-Coli.</a> Journal of Biological Chemistry. 252 (21), 7850-7861 (1977).</li><li>Crowe, J. H., Hoekstra, F. 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=Anhydrobiosis.">Anhydrobiosis.</a> Annual Review of Physiology. 54, 579-599 (1992).</li><li>Leslie, S. B., Israeli, E., 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=Trehalose+and+Sucrose+Protect+Both+Membranes+and+Proteins+in+Intact+Bacteria+during+Drying.">Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.</a> Applied and Environmental Microbiology. 61 (10), 3592-3597 (1995).</li><li>Nesarikar, V. V., Nassar, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cations+and+anions+on+glass+transition+temperatures+in+excipient+solutions.">Effect of cations and anions on glass transition temperatures in excipient solutions.</a> Pharmaceutical Development and Technology. 12 (3), 259-264 (2007).</li><li>You, Y., Ludescher, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sodium+chloride+on+molecular+mobility+in+amorphous+sucrose+detected+by+phosphorescence+from+the+triplet+probe+erythrosin+B.">The effect of sodium chloride on molecular mobility in amorphous sucrose detected by phosphorescence from the triplet probe erythrosin B.</a> Carbohydr. Res. 343 (2), 350-363 (2008).</li><li>Crowe, J. H., Hoekstra, F. A., Nguyen, K. H. N., Crowe, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+vitrification+involved+in+depression+of+the+phase+transition+temperature+in+dry+phospholipids.">Is vitrification involved in depression of the phase transition temperature in dry phospholipids.</a> Biochim. Biophys. Acta Biomembr. 1280, 187-196 (1996).</li></ol>

没有利益冲突的声明。

这项研究的动机是探讨一些配方的特性，可能是​​冷冻干燥过程中的细胞存活的重要性。虽然不同物种之间的内在干燥公差变化，如在图1A中所示，不同配方支持细胞的存活以及类似的趋势。它是信息开始，蔗糖和聚蔗糖的比较。据认为，制剂的关键因素，以支持细胞的存活能力的制剂成分（次）来代替水在脱水过程中，从而保持在干燥状态下的细胞的蛋白质和膜的结构。双糖（如...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Ficoll PM 400</td>
			<td>GE-healthcare</td>
			<td>17-0300-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEC (Natrosol-M Pharm Grade)</td>
			<td>Ashland</td>
			<td>&nbsp;</td>
			<td>Gift from Ashland</td>
		</tr>
		<tr>
			<td>HPMC (Methocel F4M)</td>
			<td>Dow</td>
			<td>&nbsp;</td>
			<td>Gift from the Department of Pharmacy, Uppsala University</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S2395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptic Soy broth</td>
			<td>Merck</td>
			<td>105459</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>Merck</td>
			<td>105458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lyostar II</td>
			<td>FTS Kinetics</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pyris Diamond DSC</td>
			<td>Perkin-Elmer</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bruker AXS SMART CCD 1k Diffractometer</td>
			<td>Bruker</td>
			<td>N.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-beam FEI Strata DB235 FIB/SEM</td>
			<td>FEI</td>
			<td>N.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kr&uuml;ss Educational Tensiometer</td>
			<td>Kr&uuml;ss</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

formulations for freeze-drying of bacteria and their influence on cell survival

细胞水，可以去除可逆性灭活微生物，以方便存储。去除的一种这样的方法是冷冻干燥，这被认为是一个温和的脱水方法。在干燥过程中，以促进细胞的存活，细胞常常预先制定。的配方形成嵌有该细胞，并保护他们免受各种有害应力，在冷冻干燥过程中施加于细胞的矩阵。在这里，我们提出了一种通用的方法来评价冷冻干燥后的细胞的存活率，我们说明通过比较所得到的结果与四个不同的配方：二糖蔗糖，蔗糖衍生的聚合物聚蔗糖PM400，和各自的多糖羟乙基纤维素（HEC ）和羟丙基甲基纤维素（HPMC），两种菌株的细菌，P.恶臭 KT2440和A. A6 chlorophenolicus。在这项工作中，我们说明如何准备冷冻干燥制剂，以及如何调查mechan主义的特征的制剂，使用差示扫描量热法（DSC），表面张力测量，X-射线分析，电子显微镜和有关这些数据传送到生存率补液后的细胞的存活。对聚合物进行选择，得到各自的多糖类似蔗糖的单体结构到不同程度。使用这种方法设置，表明聚合物可以支持细胞的存活，有效二糖制剂的某些物理性质的控制1。

表1显示数据的制剂组合物，热事件记录，可通过DSC在加热过程中的冷冻制剂，干燥样品的结构和制剂溶液的表面张力。 的Tg&#39;的蔗糖已被确定为-40°C 2，3，并可能难以检测蔗糖浓度低于20％w / w的。在-35°C的热事件可能与发病冰溶解2。在HEC和HPMC的样品的晶体结构通过X射线检测，也可见于扫描电镜（ 见图2b和2c）与正常的NaCl晶体形式重叠?...

Watch this Scientific Journal Video about Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival at JoVE.com

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

冷冻干燥往往是一个简单方便的方式来获得可行的细菌细胞的干制品。一个问题的过程，是细胞的存活。我们这里详细调查冷冻干燥过程中的细胞的存活的影响所用的公式的属性的一个过程。

冷冻干燥细菌及其影响细胞生存的配方

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

formulations-for-freeze-drying-bacteria-their-influence-on-cell

Research

JoVE Journal

Biology

23.2K 次观看.  Swedish University of Agricultural Sciences. 冷冻干燥往往是一个简单方便的方式来获得可行的细菌细胞的干制品。一个问题的过程，是细胞的存活。我们这里详细调查冷冻干燥过程中的细胞的存活的影响所用的公式的属性的一个过程。

视频: Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

获得血细胞从夏威夷短尾鱿鱼 Euprymna scolopes和观察他们坚持共生和非共生细菌

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

科研

生物学

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

检测采用ELISA法的母语完整的核小体的翻译后修饰

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

为极化上皮细胞注射质粒的小GTP酶的功能分析

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

策略跟踪Anastasis，A细胞存活的现象，细胞凋亡反转

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

生存时间的测量<em&gt;臂</em&gt;轮虫：产妇条件同步

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

用活细胞显微镜研究低压等离子体灭菌对枯草芽孢杆菌孢子存活的不利影响

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...