This article was supported by Award Number R01GM087227 from the National Institute of General Medical Sciences, the Norman Ackerman Advanced Research Program, and the Robert A. Welch foundation (Grant A-1769).

1. SPPS:CK（TMR）TATG（fTAT）合成注意:dfTAT产生于两个步骤:合成的单体fTAT通过固相肽合成接着二硫键二聚化以形成dfTAT的。 <ol><li>在一个标准的50 ml的SPPS容器溶胀二甲基甲酰胺（DMF）将500mg Rink酰胺MBHA树脂的1小时。 </li><li>通过温育的Fmoc保护的树脂中20％哌啶溶液（20％哌啶的DMF，将10毫升（0.30毫摩尔）进行Fmoc去保护。（1×5分钟和1×15分钟）用洗涤步骤使用贯彻去保护步骤两次DMF（洗涤步骤包括冲洗所述树脂多次使用的约150毫升DMF，总体积并除去通过真空过滤）中的溶剂中反应之间。 </li><li>合成CK（TMR）RKKRRQRRRG（fTAT）在Rink酰胺MBHA树脂。使用下面的Fmoc保护的氨基酸:Fmoc-赖氨酸（MTT）-OH（仅在N末端），将Fmoc-赖氨酸（BOC）-OH，使Fmoc-Gly-OH，Fmoc基 - 精氨酸（了Pbf）-OH，的Fmoc- GLN（TRT）-OH，和Fmoc-半胱氨酸（TRT）-OH。进行，使用N 2（20 PSI）以搅动该反应的反应。完成所有的反应在室温。 <ol><li>进行4小时的每个氨基酸偶联反应。对于每一个耦合，使用Fmoc基保护的氨基酸（1.2毫摩尔），N，N，N&#39;，N&#39; -四甲基-O-（1- -1H-苯并三唑 -1-基）脲六氟磷酸酯（HBTU）（0.44克，1.1毫摩尔），和二异丙基乙胺（DIEA）（0.51毫升，3.0毫摩尔）溶解在DMF中。以下的4小时耦合，用DMF洗涤树脂，并如在步骤1.2中所述进行Fmoc去保护步骤。 </li><li>重复，直到fTAT的线性肽链:合成（线性肽链CKRKKRRQRRRG没有TMR）。 </li><li>然后彻底使用第一DMF中，并以下，与二氯甲烷（DCM）洗涤树脂。冲洗树脂多次使用的约150毫升DMF或DCM的总体积与除去溶剂通过真空过滤。 </li><li> ü本身MALDI-TOF，确认获得的肽具有正确的分子量。 <ol><li>通过将1毫克矩阵到一个50微升的0.01％TFA的水溶液和50微升的乙腈组成的溶液使一个矩阵组合。 </li><li>以确认线性肽链的质量，使用α氰基-4-羟基肉桂酸。运行粗肽的分析型HPLC和从纯峰的顶部收集50微升。 </li><li>以制备样品到板在MALDI板上:添加8微升肽到2微升基质溶液和就位在MALDI板上的空气干燥或在37℃的热板。 注意:精氨酸是已知的氨基酸难以夫妇在SPPS。成功的偶合是通过执行一个Kaiser试验14（例如，茚三酮试验）确定。这种测试允许的游离胺可能留下的未偶联在树脂上的检测。万一蓝色被检测（指示游离胺的存在），耦合小号TEPS是重复的。 </li></ol></li></ol></li><li>为CK（-NH-TMR）TATG（fTAT），裂解MTT保护基赖氨酸的ε-氨基上的CK（-NH-MTT）TATG用的1％三氟乙酸（TFA）和2％三异丙基硅烷组成的溶液（TIS）的DCM。 <ol><li>作为除去MTT保护基将导致黄色的外观，孵育用20毫升上述溶液的树脂5分钟，重复上述操作直至没有黄色是观察。之间在洗净用DCM和DMF的树脂。此外，因为TIS是清除剂，将清除的MTT法，一旦无黄颜色出现在MTT法的存在下该溶液中。 </li><li>添加用1％TFA的DCM溶液（无TIS）的附加溶液的树脂，以确保没有更多的MTT是除去并将溶液保持清澈。 </li></ol></li><li>溶解的三个组成部分:Carboxytetramethylrhodamine（TMR），HBTU，和DIEA（4，3.9和10等价于相对于树脂（以mg）的量）的DMF并添加此混合物到树脂中，进行该反应O / N使用干燥的N 2，以提供搅拌。 </li><li>以下的Fmoc脱保护和氨基酸缀合，用DCM洗涤树脂，并让其干燥。用于从树脂上的肽的完全切割，添加含有92.5％TFA，2.5％的溶液H 2 O的，2.5％，TIS和2.5％乙二硫醇（EDT）（总体积= 4毫升）的肽基树脂3小时，在室温以实现全球去保护和切割从树脂。 <ol><li>由排出溶液从步骤1.6成40 ml的Et 2 O等的沉淀用冷的无水乙醚（Et 2 O等）的粗肽的产品，旋下来，在4℃和4000×g离心20 - 25分钟。重复此步骤，以允许用冷无水乙醚2 O沉淀物的洗涤</li><li>重悬沉淀物在水（5ml或直至沉淀完全溶解），并冷冻干燥。然后重悬在0.1％TFA水溶液/乙腈制得的产品。 </li></ol></li> &lt;李&gt;用分析C18柱（5微米，4×150毫米），以分析每一个肽进行HPLC分析。使用1毫升/分钟的流速，并检测在214nm和550nm处。 </li><li>在C18 10×250毫米柱的肽纯化进行半制备型HPLC。使用的4毫升/分钟的流速，并检测在214nm和550nm处。对于所有的运行时，使用0.1％TFA水溶液（溶剂A）和90％乙腈，9.9％水和0.1％TFA（溶剂B）中的线性梯度。 </li><li> fTAT，预期质量::2,041.17，观察到的质量:2040.66根据制造商的协议通过MALDI-TOF确定的肽的正确身份。 DEAC-K9，预期质量:1,412.97，观察肿块:1,415.59。使用α-氰基-4-羟基肉桂酸矩阵的MALDI-TOF。 </li></ol> 2.氧化反应:dfTAT代<ol><li>通气的磷酸盐缓冲盐水（PBS）pH 7.4的1小时（至少5毫升下面的反应）。 </li><li>在AER溶解fTAT（0.3毫克，1.5×10 -4毫摩尔）ated pH 7.4的PBS（5毫升）中。确保的pH为7.0之间 - 7.5在加入肽后。如果不添加氢氧化钠（1M，以小的增量1 - 5微升）以使pH恢复到7.4。 </li><li>注意:氧溶解在PBS作用以氧化硫醇基上fTAT和形成二硫键。 </li><li>章动反应O / N，以允许它直到完成（100％收率基于HPLC分析）反应。使用反相HPLC提纯产物和通过质谱法（MALDI-TOF）如在步骤1.9分析。预期质量:4,080.34，观察肿块:4,084.21。 </li><li>冷冻干燥纯dfTAT（0.71×10 -4毫摩尔），并悬浮在200μl水（浓度纯dfTAT = 356.3μM）。 </li></ol> 3.测量dfTAT浓度<ol><li>在149微升的50mM TCEP溶液重悬纯化dfTAT（通常为1微升取决于肽的纯化的量）的等分试样。 </li><li>注:在这一步dfTAT减记至莫nomer对口fTAT消除时出现的吸光度淬火由于TMR荧光团在dfTAT近距离（TMR在dfTAT消光系数ε减小相比TMR的降低fTAT的ε）。 </li><li>允许样品大约20分钟反应（分析性HPLC可用于确认形成fTAT的）。 </li><li>添加所有的溶液到石英比色皿，测量吸光度556纳米。 </li><li>使用比尔定律（A =εcl:ε= 91500 M-1 -1）来计算fTAT的浓度，确定dfTAT的浓度，并划分[fTAT]由两个。 </li></ol> 4.蜂窝交货实验<ol><li>种子在培养皿中的细胞（HeLa细胞，HDF， 等）在80汇合- 90％（例如，8孔或24孔皿）。生长的细胞在适当的培养基（例如，DMEM补充有10％FBS和青霉素/链霉素），直到80 -在37℃90％汇合湿润气氛含有5％ 的 CO 2。 </li><li> （通过添加200微升PBS，然后除去它三次）洗涤细胞，用PBS三次（3倍）。 </li><li>使dfTAT的5μM的工作浓度在NRL-15培养基稀释dfTAT（在水中）的股票（对于一个8井菜的总体积应为200微升）。 5微米dfTAT甲浓度导致有效递送在测试日期（ 图3）大多数细胞类型（在90％以上的细胞中存在的碟形高水平胞质递送）。然而，较低或较高的浓度可能是更充分的用于与高或低的倾向为渗透的细胞类型。 注意有关介质:NRL-15用于dfTAT的交付，因为它不含有半胱氨酸可能降低dfTAT二硫键。然而，我们的数据表明，这两个定期L-15（半胱氨酸）和DMEM可以用作传递介质。 L-15的包含还原氨基酸半胱氨酸但半胱氨酸presumabLY氧化在媒体上形成胱氨酸（DMEM是制定与胱氨酸）。因此dfTAT丝毫无损这些媒体和传送工作在相同的效率，与nrL15获得。 </li><li>孵育细胞dfTAT（5μM）具有或不具有货物（例如，绿色荧光蛋白（10μM）），并保持在37℃下进行1小时（温育时间可以减少，但dfTAT通常需要大约30至45分钟，以诱导内体泄漏）。 </li><li>用肝素（1毫克/毫升）中的L-15洗涤细胞（3次洗涤，建议）以除去dfTAT结合于细胞的质膜上。 </li><li>孵育细胞与细胞不透核染色剂，例如，SYTOX蓝，SYTOX绿（2μM在NRL-15），以确定细胞的质膜是否受到损害，根据（死细胞将同时活细胞不会被染色）制造商的协议。 </li><li>使用荧光显微镜（100X油浸或20X物镜）图像的细胞。使用RFP滤波器（图片dfTAT防爆=560±20纳米/ EM = 630±35纳米）。 注意:成功递送导致dfTAT的漫荧光整个细胞（评估蛋白质或感兴趣的肽的递送将取决于应用）。核仁由fTAT（dfTAT的时胞质条目的还原产物）的染色可以用作一个指示检测到的荧光是细胞内的。 fTAT将在几个小时内降解。在这一点上，降解片段的荧光将显示为点状。这不应该混淆为点状分布也可以看出，如果dfTAT仍然不成功内内体困（这可能发生，如果dfTAT出现在太低的浓度）。 </li></ol> MOI 5. Controling集中交付<ol><li>确定实现dfTAT中使用的细胞类型高效胞浆释放所需要的"最优配送"的浓度。在细胞培养与increasin执行荧光显微镜克浓度dfTAT的。最佳dfTAT浓度被定义为最小浓度，导致清除胞质"扩散"TMR荧光细胞的大约100％在培养。 </li><li>改变MOI的浓度用在共温育协议，同时保持dfTAT恒定的浓度（例如，使用dfTAT的最佳递送浓度）。改变培养时间以不足1小时也可以是一种选择，以改变的量的MOI递送。 </li></ol>

<ol>
	<li>Didenko, V. V., Ngo, H., Baskin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethyleneimine+as+a+transmembrane+carrier+of+fluorescently+labeled+proteins+and+antibodies.">Polyethyleneimine as a transmembrane carrier of fluorescently labeled proteins and antibodies.</a> Anal Biochem. 344, 168-173 (2005).</li><li>Takeuchi, 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=Direct+and+Rapid+Cytosolic+Delivery+Using+Cell-Penetrating+Peptides+Mediated+by+Pyrenebutyrate.">Direct and Rapid Cytosolic Delivery Using Cell-Penetrating Peptides Mediated by Pyrenebutyrate.</a> ACS Chem Biol. 1, 299-303 (2006).</li><li>Morris, M. C., Depollier, J., Mery, J., Heitz, F., Divita, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peptide+carrier+for+the+delivery+of+biologically+active+proteins+into+mammalian+cells.">A peptide carrier for the delivery of biologically active proteins into mammalian cells.</a> Nat Biotech. 19, 1173-1176 (2001).</li><li>Zhang, H., 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=Reprogramming+of+somatic+cells+via+TAT-mediated+protein+transduction+of+recombinant+factors.">Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors.</a> Biomaterials. 33, 5047-5055 (2012).</li><li>Torchilin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+delivery+of+protein+and+peptide+therapeutics.">Intracellular delivery of protein and peptide therapeutics.</a> Drug Discov Today: Technol. 5, e95-e103 (2005).</li><li>Chakravarty, P., Qian, W., El-Sayed, M. A., Prausnitz, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+molecules+into+cells+using+carbon+nanoparticles+activated+by+femtosecond+laser+pulses.">Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses.</a> Nature nanotechnol. 5, 607-611 (2010).</li><li>Kaczmarczyk, S. J., Sitaraman, K., Young, H. A., Hughes, S. H., Chatterjee, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+delivery+using+engineered+virus-like+particles.">Protein delivery using engineered virus-like particles.</a> Proc Natl Acad Sci U S A. 108, 16998-17003 (2011).</li><li>Pan, C., Lu, B., Chen, H., Bishop, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+human+fibroblasts+using+HIV-1+TAT+recombinant+proteins+OCT4,+SOX2,+KLF4+and+c-MYC.">Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC.</a> Mol Biol Rep. 37, 2117-2124 (2010).</li><li>Fittipaldi, 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=Cell+Membrane+Lipid+Rafts+Mediate+Caveolar+Endocytosis+of+HIV-1+Tat+Fusion+Proteins.">Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins.</a> J Biol Chem. 278, 34141-34149 (2003).</li><li>Lee, Y. J., Erazo-Oliveras, A., Pellois, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+macromolecules+into+live+cells+by+simple+co-incubation+with+a+peptide.">Delivery of macromolecules into live cells by simple co-incubation with a peptide.</a> Chembiochem. 11, 325-330 (2010).</li><li>Angeles-Boza, A. M., Erazo-Oliveras, A., Lee, Y. J., Pellois, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+endosomolytic+reagents+by+branching+of+cell-penetrating+peptides:+tools+for+the+delivery+of+bioactive+compounds+to+live+cells+in+cis+or+trans.">Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans.</a> Bioconjugate chem. 21, 2164-2167 (2010).</li><li>Walev, I., 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=Delivery+of+proteins+into+living+cells+by+reversible+membrane+permeabilization+with+streptolysin-O.">Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.</a> Proc Natl Acad Sci U S A. 98, 3185-3190 (2001).</li><li>Erazo-Oliveras, 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=Protein+delivery+into+live+cells+by+incubation+with+an+endosomolytic+agent.">Protein delivery into live cells by incubation with an endosomolytic agent.</a> Nat methods. 11, 861-867 (2014).</li><li>Kaiser, E., Colescott, R. L., Bossinger, C. D., Cook, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+test+for+detection+of+free+terminal+amino+groups+in+the+solid-phase+synthesis+of+peptides.">Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides.</a> Anal Biochem. 34, 595-598 (1970).</li><li>Muthukrishnan, N., Johnson, G. A., Lim, J., Simanek, E. E., Pellois, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAT-mediated+photochemical+internalization+results+in+cell+killing+by+causing+the+release+of+calcium+into+the+cytosol+of+cells.">TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells.</a> Biochim biophys acta. 1820, 1734-1743 (2012).</li><li>Lautwein, 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=Human+B+lymphoblastoid+cells+contain+distinct+patterns+of+cathepsin+activity+in+endocytic+compartments+and+regulate+MHC+class+II+transport+in+a+cathepsin+S-independent+manner.">Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner.</a> J Leukoc Biol. 75, 844-855 (2004).</li></ol>

The authors have nothing to disclose.

因为这用于dfTAT交付不应过分融合的细胞（&gt; 90％汇合）可能会影响传递效率。该细胞也应该是健康的:死细胞的培养可以释放凋亡片段与dfTAT可交互（例如，从DNA降解核）。这反过来又可以与递送效率和成像质量干扰。细胞应彻底洗净加入dfTAT之前删除FBS。 BSA存在的FBS可以结合dfTAT并且这可以降低dfTAT的递送效率。洗涤然而，应当小心以过大的力进行可能导致贴壁细胞从培养皿分离或引起的应力，其导致较低的内吞uptake.When递送蛋白质/肽使用dfTAT，蛋白质/肽储备液样品应足够浓缩避免过度稀释夺标，15家媒体的孵化过程中: 例如，将在5 - 10μL样品到200μ升NRL-15的建议。 荧光标记的细胞穿透肽可以显示一个光诱导膜破坏活性。15这是当高强度光剂量用于dfTAT的情况。它可通过细胞内细胞器（例如，内体，线粒体），细胞表面起泡，和细胞死亡的破裂表现出来。为了尽量减少这些影响，应注意，以保持光曝光到最低限度（通过共聚焦或落射荧光通常耐受成像所需的标准条件）。 当递送MOI如蛋白质，一种是面对于每一个大分子是唯一的，并且，因此，每一个输送实验可能需要微调（远远超过在DNA转染的，其中的分子递送的情况下始终是带负电荷的问题发A，T，G和C聚合物）。故障排除因此，重要的几个方面应予以考虑。首先，具有极低的电点蛋白质可静电结合dfTAT和灭活它。与此相反，具有非常高的电点的蛋白质可能与dfTAT竞争与细胞表面上的带负电荷的葡萄糖胺聚糖结合。这可能进而降低细胞内吞作用和减少低于核内体溶解的阈值的吸收。在这两种情况下，一个可能的解决这个问题的不断增加dfTAT浓度。 正如预期的那样，由于细胞蛋白酶的存在（如组织蛋白酶16）沿内吞途径，内务部分娩过程中的降解是一个问题。而dfTAT可以提供完整的蛋白质，尚未建立蛋白质的递送过程中被完全或部分降解的量。这又是一个问题，是MOI相关的，且应根据所追求的应用进行监测。 dfTAT是一种高效的投递代理。为dfTAT的活动REMA的分子基础然而，移民局目前还不清楚。具体地，所需要的实现内体逃逸的结构或化学特征尚未确定。因此，现在无法预测多少dfTAT的结构可以不改变递送效率进行修改。我们已经确定，存在于dfTAT二硫键可以通过非还原的连接体代替，而不会有害作用。附加构效关系正在逐步建立之中。 我们的数据表明，有可能降低该肽的孵育时间小于1小时（低到5分钟已经执行）。然而，dfTAT释放细胞内人口众多的细胞没有观察到，直到大约15-30分钟。这表明，短的温育时间可以是足以dfTAT内吞作用或细胞摄取。然而，需要您需要从内吞途径dfTAT逃逸内体成熟。 <p类="jove_content"&gt;最后，我们已经建立了多个MOI可以传送一次（使用本文所呈现的协议，用一个以上的MOI的期间共培养中使用的唯一的例外）。此外，dfTAT介导的递送可以重复（交付步骤重复20分钟分开已经过测试）。这并不影响细胞生存力。因此，我们认为，本文提出的传输协议是非常方便，高效，无害的细胞。

递送蛋白质，肽或细胞不可渗透的小分子进入活细胞是在许多生物或生物技术应用（细胞成像，功能测定法，细胞重新编程等 ）1-4常常合乎需要的。许多递送方法已被报道，包括显微注射，电穿孔，或用载体试剂（例如，细胞穿透肽，例如TAT，脂质）5-7。每种技术一般都有特定的优点和缺点，可能使这些方法足以满足一定的应用，但不是为别人。常见问题涉及在少数细胞而不在整个贫困人口递送效率和/或控制的多少材料输送8,9，毒性或有害生理影响10,11，缺乏临时控制的，交付的缺乏（例如，显微注射）12，和复杂的化学缀合或制剂方案11。 S ="jove_content"&gt;最近，我们已经开发了一种新的交付策略绕开这些限制。这一战略依赖于一个名为dfTAT肽（二聚体荧光TAT）13。 dfTAT从公知的细胞穿透肽（CPP）的TAT而得。 dfTAT包含TAT的两个二硫键结合拷贝标记荧光团四甲基罗丹明。尽管他们的相似之处，TAT和dfTAT活性显著不同。 TAT是典型的内吞作用内化进入细胞。这CPP仍然但是在大多数被困在内涵体（这通常会导致细胞内的肽的点状分布时，利用荧光显微镜检查）。像TAT，dfTAT有效地被细胞内吞。然而，dfTAT不会留在里面内涵体困。相反，它介导内体泄漏的方式，是非常有效的。 dfTAT的内体裂解活性可以然后被利用通过一个简单孵育测定递送大分子。 ntent"&gt;传送过程的当前理解如下。dfTAT诱导巨吞。其结果是，细胞孵育dfTAT占用的可溶性蛋白，肽或小分子（感兴趣的分子，MOI）存在于介质通过流体相内吞作用（ 参见图1）。dfTAT和MOI之间的相互作用不是必须的，只要内吞途径中一起两个实体通信。正如dfTAT到达内吞细胞器的内腔中一个特定的阈值，它表示它的内体泄漏活性（的分子细节仍有待充分表征的）。漏细胞器的内腔的内容，因此，教学语言，然后被释放到细胞中。因此，这种方法是与MOI没有缀合或制剂计划必须很方便的。此外，由于dfTAT是不直接修改一个MOI，它还应不同的MOI功能干扰一旦细胞内递送的实现。此外，浓度dfTAT用于交付是独立于MOI的媒体。例如，dfTAT浓度可以保持不同的实验之间保持恒定，以保证在内体泄漏重现的效率。与此相反，MOI在媒体浓度可逐渐改变以达到所需水平的细胞质MOI交付。 实现了与dfTAT高内体泄漏效率显着地无害的许多测试日期的细胞。这是一个惊喜，因为内吞细胞器是细胞的重要组成部分，人们所期望的显着泄漏通过dfTAT介会伴随着有害的细胞反应。然而，处理的细胞增殖以相同的速率作为未经处理的细胞，并且不显示在其转录任何显著改变。此外，可交付使用可重复的递送效率分钟内反复，这表明细胞可以容忍或恢复从分娩过程，而不会丢失他们的能力，内吞作用或内体泄漏。 dfTAT交付和什么这些反应可能是有待探索的分子细节中微妙的细胞反应可能发生。然而，通过结合高效率，便利性的协议，并没有毒性，这种交付方法应该证明在许多细胞为基础的应用可以立即投入使用。本文提出的协议的目的是使该技术访问研究界。

<table><tbody><tr><td>Heparin&amp;nbsp;</td><td>Sigma</td><td>CAS 9041-08-1</td><td></td></tr><tr><td>SYTOX Blue</td><td>Invitrogen</td><td>S11348</td><td></td></tr><tr><td>SYTOX Green</td><td>Invitrogen</td><td>S7020</td><td></td></tr><tr><td>nrL15 L-15 (&amp;ndash;) cysteine</td><td>Hyclone</td><td></td><td>Special order</td></tr><tr><td>Fmoc-protected Amino acids</td><td>Novabiochem</td><td></td><td></td></tr><tr><td>dfTAT</td><td></td><td></td><td>Samples will be provided upon request (contact: pellois@tamu.edu)</td></tr><tr><td>Biosafety Cabinet</td><td></td><td></td><td></td></tr><tr><td>Inverted epifluorescence microscope</td><td>Olympus</td><td>model IXB1</td><td>equipped with a heating stage maintained at 37 &amp;deg;C and with a Rolera-MGI Plus back-illuminated electron-multiplying charge-coupled device (EMCCD) camera (Qimaging).&amp;nbsp;</td></tr><tr><td>37 &amp;deg;C humidified, 5% CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td></td><td></td><td></td></tr><tr><td>Peptide synthesizer or vessel to preform manual peptide synthesis</td><td></td><td></td><td></td></tr></tbody></table>

delivery of proteins, peptides or cell-impermeable small molecules into live cells by incubation with the endosomolytic reagent dftat

大分子递送策略通常利用内吞途径蜂窝条目的路由。然而，内体截留严重限制与大分子穿透细胞的胞质空间的效率。最近，我们通过确定试剂dfTAT，标记有荧光团四甲肽TAT的二硫键二聚体规避这个问题。我们已经生成原型细胞穿透肽（CPP）的TAT，dfTAT，穿透活细胞和到达细胞具有特别高的效率的胞质空间的荧光标记的二聚体。胞质递送dfTAT的是在多种细胞系，包括原代细胞来​​实现的。此外，交货不会明显影响细胞的活力，增殖或基因表达。 dfTAT可以提供小分子，肽，抗体，生物活性酶和转录因子。在这份报告中，我们描述参与DFTA协议T合成和细胞输送。手稿描述如何通过改变蛋白质的细胞外给药量以控制蛋白质递送至细胞的胞质空间的量。最后，这种新技术和涉及验证输送步骤的当前局限性进行了讨论。所描述的方案应为基于细胞的测定以及对于离体操作的细胞和重编程是非常有用的。

为了评估fTAT和dfTAT之间的差，HeLa细胞被温育1小时以每种肽，以确定在他们的细胞定位的差异。使用荧光显微镜的两个CPP的内化进行了评估。 图2表明，fTAT（20μM）定位于点状分布。这种分布是一致的肽剩余的夹带里面内涵体。与此相反，dfTAT（5微米）的荧光信号显示整个胞质溶胶和细胞核的均匀分布;图3示出了 dfTAT的胞质分布在许多不同的细胞系，包括COLO 316，NIH 3T3，将HaCaT，被观察到的难以转染神经2a中，MCH58的初级细胞系的HDF，DRG-F11和NCL-H1299。 20倍的图像显示了一个非常高的比例（&gt; 80％），在培养皿中显示dfTAT的胞浆分布，无细胞毒性（无SYTOX蓝核staininG）。 为了确定dfTAT介导的内体泄漏是否可以提供大量的蛋白质进入细胞的细胞质。 EGFP（26 kDa的）被选择作为模型蛋白质。这是因为它的荧光可以使用通过观察绿色荧光的定位（如果正确折叠）来检测其输送到细胞中。要做到这一点测定中，EGFP和政变孵育1小时以细胞， 如图4，EGFP显示的胞质和类似于所观察到的dfTAT中超过90％的细胞而没有明显毒性核绿色荧光分布。 <img alt="图1" src="/files/ftp_upload/53175/53175fig1.jpg" /> 的dfTAT介导分子传递图1.示意图显示原理 。由左到右。示意图显示dfTAT细胞传递以及一个细胞防渗货物。第一dfTAT诱导的内吞作用这导致uptakdfTAT电子随货物进入内吞小泡。在第二步骤dfTAT逸出从吞囊泡这导致dfTAT的释放和细胞不可渗透的分子进入细胞的胞质溶胶中。哺乳动物细胞的细胞质是还原环境，因此在细胞质dfTAT减少到它的单体对应fTAT。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53175/53175fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53175/53175fig2.jpg" /> 这两种HeLa细胞孵育使用100X目的。fTAT单色图像显示的细胞表现出荧光点状分布，而dfTAT图像显示单元20μMfTAT（左图）和5微米dfTAT（右面板）图2.荧光和明视野图像显示均匀的胞质和核fluorescenc<html> Ë分布。比例尺:10微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53175/53175fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53175/53175fig3.jpg" /> 图3.高效的交付dfTAT进入活细胞达到在多种细胞系 。测试的细胞系如下:HeLa细胞，NIH 3T3，COLO 316和角质形成，神经2A，MCH58，HDF，DRG-F11和NCL- H1299。细胞与5μM的dfTAT 1小时和之后的洗涤步骤，根据协议和成像。检测出的荧光信号是在细胞质和细胞的核（顶面板:20X物镜，底部面板:100X物镜）。所述细胞不透过性核染色剂SYTOX蓝用于dfTAT处理后，以评估细胞生存力。比例尺，20X目标:50微米; 100X目标:10微米。jove.com/files/ftp_upload/53175/53175fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53175/53175fig4.jpg" /> 图4. dfTAT推出完整的EGFP成不同的细胞系。（A）中的HeLa（上图）和NIH 3T3（底部面板）细胞与EGFP（10μM）和dfTAT（5μM）1小时，并随后的步骤进行根据协议。图像显示EGFP的两种细胞系中的均匀胞质荧光分布。比例尺为10μm。（B）的 HeLa和初级HDF细胞与EGFP（10μM）和dfTAT（5μM）根据协议。 20X图像显示EGFP和dfTAT在HeLa和初级HDF细胞的均匀胞质荧光分布。覆盖（伪:白色）表示dfTAT存在（伪:紫色），绿色荧光蛋白（pseudocol或:绿色）均HeLa和HDF细胞的胞质空间。 SYTOX蓝（蓝色示出）被用来驴细胞活力。比例尺:50微米<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53175/53175fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT at JoVE.com

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

delivery-proteins-peptides-or-cell-impermeable-small-molecules-into

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

10.2K 次观看.  Texas A&M University. We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky. 

视频: Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

生产二硫键稳定的跨膜肽复合物的结构研究

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

科研

化学

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

生物工程

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

生物化学

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

免疫与感染

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

工程细胞透水蛋白

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

生物学

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

测量肽转运到大Unilamellar囊泡

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

蛋白质直接送货到哺乳动物细胞利用细胞渗透半胱氨酸<sub&gt; 2</sub&gt; -His<sub&gt; 2</sub&gt;锌指域

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

成像 FITC-葡聚糖作为胞吐作用调节的记者

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...

Bead Loading Proteins and Nucleic Acids into Adherent Human Cells

Many live-cell imaging experiments use exogenous particles (e.g., peptides, antibodies, beads) to label or function within cells. However, introducing proteins into a cell across its membrane is difficult. The limited selection of current methods struggles with low efficiency, requires expensive and technically demanding equipment, or functions within narrow parameters. Here, we describe a relatively simple and cost-effective technique for loading DNA, RNA, and proteins into live human cells. Bead loading induces a temporary mechanical disruption to the cell membrane, allowing macromolecules to enter adherent, live mammalian cells. At less than 0.01 USD per experiment, bead loading is the least expensive cell loading method available. Moreover, bead loading does not substantially stress cells or impact their viability or proliferation. This manuscript describes the steps of the bead loading procedure, adaptations, variations, and technical limitations. This methodology is especially suited for live-cell imaging but provides a practical solution for other applications requiring the introduction of proteins, beads, RNA, or plasmids into living, adherent mammalian cells.

Bead loading introduces proteins, plasmids, and particles into adherent mammalian cells. This cell loading technique is inexpensive, rapid, and does not substantially affect cell health. It is best suited for live-cell imaging.

珠子将蛋白质和核酸加载到附体人类细胞中

Many live-cell imaging experiments use exogenous particles (e.g., peptides, antibodies, beads) to label or function within cells. However, introducing ...

Live Fluorescence, Inverse Imaging of Cell Ruffling, and Macropinocytosis

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase nutrients and other material in cells. The dramatic extension of cell surface ruffles, their closure to form macropinosomes, and the maturation of internalized macropinosomes are key events in this pathway that can be difficult to capture using conventional confocal imaging based on tracking a bolus of fluorescent cargo. Fluorescent dextrans are commonly used experimentally as fluid phase markers for macropinosomes and for other endocytic pathways. A method the lab has adopted to optimize the imaging of dextran uptake involves using live imaging of cells bathed in high concentrations of fluorescent dextran in the medium, with the unlabeled cells appearing in relief (as black). The cell ruffles are highlighted to visualize ruffle closure, and internalized macropinosomes appear as fluorescent vacuoles in the cell interior. This method is optimal for visualizing macropinosome features and allows for easy segmentation and quantification. This paper describes dual-labeling of pathways with different sized dextrans and the co-expression of lipid probes and fluorescent membrane proteins to demark macropinosomes and other endosomes. The detection of internalized dextran at an ultrastructural level using correlative light and electron microscopy (CLEM) is also demonstrated. These cell processes can be imaged using multiple live imaging modalities, including in 3D. Taken together, these approaches optimize macropinosome imaging for many different settings and experimental systems.

<meta charset="utf8"></head><body>活荧光、细胞褶皱的逆向成像和巨胞饮作用

This protocol demonstrates dextran imaging in live cells using continuous uptake and inverse images to optimize visualization of ruffling, macropinosome maturation, and analysis of dextran and other cell labelings.

活荧光、细胞褶皱的逆向成像和巨胞饮作用

Macropinocytosis is a highly conserved but still incompletely understood process that is essential for the uptake and ingestion of fluid, fluid-phase ...