这项工作是由美国国家卫生研究院，美国国立糖尿病，消化和肾脏疾病，2R01DK59558（GN）的资助。 UAMS翻译研究所流式细胞仪核心提供了部分资金支持由国立卫生国家研究资源中心授予UL1 RR029884在UAMS。我们感谢沛沛平博士（加州大学洛杉矶分校，洛杉矶，CA）提供的一份腺病毒携带基因编码的显性负（无效）突变体的PKC-ε和，艾伦博士Samarel（Loyola大学医学中心;梅伍德，IL），用于提供一个等分PKC-ε的组成型活性的突变体的腺病毒载体编码。我们也感谢博士。彼得·帕克和彼得·萨格登（伦敦帝国学院，伦敦，英国）组成性激活PKC-ε的cDNA编码。

1。原代培养肾小管上皮分离<ol><li>麻醉兔，切除双侧肾脏，并将其放置在培养皿中充满了消毒缓冲（DMEM/F12介质）在层流罩。 </li><li>每个肾灌注用50ml准备缓冲后跟50毫升无菌磷酸盐缓冲盐水，pH 7.4（PBS）中，PBS-氧化铁溶液（45毫升+ 5毫升）。德封装的肾脏和转移他们的准备缓冲液去铁胺。 </li><li>收获皮质每个肾。 </li><li>使用15毫升的Dounce匀浆器匀化组织。超过2无菌网筛（85微米的底部和顶部235微米的）放置超过1升的无菌烧杯中倒入匀浆。冲洗筛去铁胺的缓冲。 </li><li>收集85μm的筛上留下的任何组织，在无菌的离心管中填充用35-40毫升的去铁胺的含缓冲。轻轻混合细胞悬浮液。 </li><li>建立一个强大米agnet管外部的除去铁填充肾小球，让悬浮液中定居。从磁铁使用巴斯德吸管的吸液铁。重复此过程。 </li><li>准备1毫升消化培养基含有2,400-2,500单位的I型胶原酶和0.6毫克的大豆胰蛋白酶抑制剂溶解在去铁胺的含缓冲。过滤消毒的消化介质。 </li><li>调整细胞悬浮液的体积至40ml，并加入1毫升的消化培养基。轻轻悬浮液混合17分钟，离心，在50×g离心2分钟，在4℃，在室温下孵育，吸出培养基，并添加新鲜的去铁胺缓冲液。 </li><li>重复的铁除去与磁铁的过程几次，旋转的悬浮液再次向下，吸出培养基，并加入46毫升的不含有葡萄糖的培养基中。 </li><li>轻轻混匀，并测量出500微升该悬浮液分为两个预先称重的Eppendorf管中。旋转细胞以10,000 xg 1分钟，抽吸介质，重量的颗粒。计算湿质量和蛋白质的总收率。 </li><li>板的细胞在无菌的35毫米的培养皿中，在1毫克蛋白质/培养皿，使用无葡萄糖DMEM/F12培养基的密度。在2ml无葡萄糖的DMEM/F12培养基总是在轨道摇床中在37℃下（95％空气/ 5％CO 2）的培养箱。1,2的培养细胞</li></ol> 2。肾小管上皮细胞培养<ol><li>培养基是DMEM和Ham氏F-12营养混合不加酚红，丙酮酸盐，和葡萄糖的50:50混合物，补充有15毫摩尔的NaHCO 3，15mM的HEPES，和6mM乳酸（pH值7.4，295 mosmol / KGH 2 O）。媒体补充与L-抗坏血酸-2 - 磷酸（50μM），牛胰岛素（10纳米），人转铁蛋白（5毫克/毫升），氢化可的松（50纳米），和硒（5纳克/毫升），在紧接每日媒体的变化。 </li><li>吸旧媒体使用无菌技术菜肴。温暖，新鲜的培养基中加入2毫升每道菜都使用无菌技术。重新NAL近端肾小管上皮细胞（RPTC）达到汇合后约5-6天，在电镀1,2。 </li></ol> 3。腺病毒感染的RPTC <ol><li>腺病毒感染进行融合，静态的文化RPTC后的每日媒体的变化。显性负的PKC-ε突变（dnPKC-ε），通过更换构成：1）赖氨酸与精氨酸，在436位的ATP-结合位点，以及2）在位置与谷氨酸丙氨酸159的假底域。这些突变破坏结构的激酶活性不改变PKC-ε的活性构象。3 PKC-ε组成删除其抑制假底域的残基154-163。 </li><li>添加存货溶液重组腺病毒caPKC-ε的cDNA或dnPKC-εcDNA的每个培养皿中，以获得期望的多重性感染（MOI）导致显着增加的蛋白激酶C-＆epsil的“;蛋白水平。孵育24小时腺病毒RPTC。改变媒体和培养细胞中的另一个24小时。显著磷酸化和总的PKC-ε蛋白水平的提高，可以得到使用25-50 MOI的腺病毒载体编码caPKCε。大MOI的结果增加更大的积极PKC-ε的水平，但它不建议在RPTC由于快速的细胞死亡和损失。显著水平升高的非活动的PKC-ε蛋白可以使用50-100 MOI方式获得不产生不利影响的情况下在RPTC 5 </li><li>在感染后的48小时内，抽吸介质，洗单分子膜与PBS，并收集细胞进行免疫印迹分析，以确定蛋白质水平的PKC-ε。 </li></ol> 4。 RPTC呼吸的测量<ol><li>准备一个测定缓冲液，用于测定状态3呼吸（120 mM KCl中，5mM的KH 2 PO 4，10mM的HEPES，1mM的用MgSO 4，和2mM EGTA，pH值7.4）。为了测量复杂的I耦合状态3呼吸，补充的测定缓冲液，5mM的谷氨酸，5mM的苹果酸盐和0.1毫克/毫升的毛地黄皂苷。对于复杂的II-耦合状态3呼吸，补充的测定缓冲液中的10mM琥珀酸盐，0.1μM鱼藤酮，和0.1毫克/毫升毛地黄皂苷。对于复杂的IV-耦合状态3呼吸，补充试验缓冲液，1mM的抗坏血酸盐，1mM的N，N，N&#39;，N&#39;-四甲基 - 对 - 苯二胺（TMPD），和0.1毫克/毫升毛地黄皂苷。在37℃水浴中放置的解决方案。 </li><li>吸媒体从培养皿中包含RPTC单层，加入2毫升的热态3呼吸缓冲，轻轻地刮细胞的菜用橡胶警察，将暂停Clark型电极消耗氧气室配备。 </li><li>测量状态的呼吸（ADP的情况下）。启动状态3呼吸由ADP添加至终浓度为0.4mM。由增加的最终concentr寡启动状态4呼吸通报BULLETIN为0.5微克/毫升。非耦合测量呼吸，加入0.5μMFCCP，细胞呼吸作用状态3。6,7 </li><li>从腔室中收集的细胞悬浮液，沉淀蛋白质，使用高氯酸，和自旋向下的沉淀。确定在蜂窝沉淀的蛋白质浓度。 </li></ol> 5。分析线粒体膜电位（ΔΨm）的<ol><li>准备0.5mM的JC-1溶液中的无菌培养介质中。 </li><li>缓慢加入20微升的JC-1溶液每个培养皿中，得到最终浓度为5μM。涡流盘来彻底混合染料。返回的菜肴的培养箱30分钟。 </li><li>放菜在冰上，吸出物的介质，并用冰冷的PBS洗单层两次。吸出PBS，加1毫升的新鲜PBS，刮细胞的菜，然后将其转移到离心管中。吹打起来，并成单细胞单分子膜。 </li><li>旋转暂停2分钟，ASP在1000 XG发怒的上清液，加入1毫升的PBS中，以沉淀和重新悬浮，得到单细胞悬浮液。再次重复此步骤，如果必要的话。 </li><li>旋转细胞在1000×g离心2分钟，吸取上清液，加0.5毫升的PBS，重悬颗粒。分析通过流式细胞仪检测488 nm氩离子激光激发荧光。在两个独立的通道，FL1为525 nm和590 nm的FL2在阅读排放。线粒体膜电位作为荧光比例FL2/FL1。6表示</li></ol> 6。线粒体分离<ol><li>准备隔离缓冲液：缓冲液A（10mM的HEPES，225 mM的甘露糖醇，75 mM蔗糖，0.1mM的EGTA，pH值7.4），缓冲液B（缓冲液A含有蛋白酶抑制剂，1mM的的Na 3 VO 4，1mM的氟化钠），和缓冲器C（10mM的HEPES，395 mM蔗糖，0.1mM的EGTA，pH值7.4）。在冰上执行所有步骤。 </li><li> RPTC单层洗净两次，用冰冷的PBS缓冲液。刮Eppendorf管中旋转的单分子膜在500×g离心5分钟。在1ml的缓冲液A洗涤沉淀</li><li>重新悬浮沉淀在1ml的缓冲液B中，并且该悬浮液转移到一个2毫升的Dounce匀浆器。 </li><li>均质的Dounce匀浆器中的细胞匀浆中，直到大部分的细胞在显微镜下检查时，被破坏。 </li><li>以1,000 xg离心5分钟，在4℃下离心匀浆收集上清液并和旋转下降，以15,000 xg离心10分钟，在4℃下</li><li>丢弃上清液，并重新悬浮的颗粒含有粗线粒体在1ml的缓冲液C.附带上清液以15,000 xg离心10分钟，在4℃下重复5的粒料的2次以上的洗涤。 </li><li>为了测量呼吸配合物的活动，重新悬浮线粒体沉淀在50-100微升测定缓冲液并冷冻在液氮中。慢慢地在冰上解冻样品。 </li></ol> 7。测量活动的NADH-泛醌氧化还原酶（复合体I） <ol><li>准备的测定缓冲液：10mM的KH 2 PO 4，5毫摩尔的MgCl 2，pH值7.2。加入无脂肪酸BSA和NADH，得到最终浓度为0.25％和0.25mM，分别。抗霉素A溶液（1毫克/毫升）中加入2μl至终浓度为2微克/毫升。 </li><li>在48孔透明板运行的样品。包括空白样品，有所有增加，但没有线粒体。向每孔加入500微升测定缓冲液与加法和线粒体中，并混合5分钟，在30℃下孵育板。 </li><li>启动反应，向每孔加入10微升3.25 mM的泛醌。记录的吸光度（340 nm）的为3分钟，在20秒的间隔。加入5μl的鱼藤酮溶液（1毫克/毫升）的每个孔中，并记录额外的2分钟的吸光度。计算复杂，我鱼藤酮敏感的NADH氧化活性。 </li></ol> 8。测量活动琥珀酸泛醌氧化还原酶（复合物Ⅱ） <oL&gt; <li>准备的测定缓冲液含有0.25％脂肪酸游离BSA，的20mM钾琥珀酸，0.25毫摩尔2,6 - 二氯靛酚，2微克/毫升抗霉素A，和10μg/ ml的鱼藤酮。 </li><li>在48孔透明板，包括空白样品，有所有增加，但没有线粒体，运行的样品一式两份。向每孔加入500微升测定缓冲液与加法和线粒体中，并混合2分钟，在30℃下孵育板。 </li><li>启动反应，向每孔加入10微升3.25 mM的泛醌。记录的吸光度（595 nm）的为3分钟，在20秒的间隔。计算复合物II活性的2,6 -二氯靛酚还原后。 </li></ol> 9。辅酶Q-细胞色素C还原酶活性的测量（情结III） <ol><li>准备的测定缓冲液，含有0.1％无脂肪酸的牛血清白蛋白，80mM的琥珀酸钾，10μg/ ml的鱼藤酮，和0.24 mM的钾氰化物。 </li><li>运行样品在48孔透明板，包括空白样品，有所有增加，但没有线粒体的副本。向每孔加入291微升测定缓冲液与加法和线粒体中，并混合5分钟，在30℃下孵育板。 </li><li>开始反应6 mM的氧化中的细胞色素c在各孔中加入3微升。记录的吸光度（550 nm）的为3分钟，在20秒的间隔。抗霉素A溶液（1毫克/毫升），每孔加入5μl和记录额外的2分钟的吸光度。计算复杂III活性，抗霉素A敏感的细胞色素C还原。7 </li></ol> 10。细胞色素氧化酶的活性测定（复杂IV） <ol><li>准备9的细胞色素c的1mM溶液中加入抗坏血酸钠和透析过夜，4℃下在1升的磷酸盐缓冲液中（10mM的KH 2 PO 4，pH 7.2）中含有5mM MgCl 2的溶液，降低了对细胞色素c。准备在测定缓冲液中，其中包含0.1％无脂肪酸的牛血清白蛋白和抗霉素A（2微克/毫升）。 </li><li>在48孔透明板，包括空白样品，有所有增加，但没有线粒体，运行样本。向每孔加入233微升测定缓冲液与加法和线粒体中，并混合5分钟，在30℃下孵育板。 </li><li>除了减少，细胞色素c（90μM的最终浓度），开始反应。记录的吸光度（550 nm）的为3分钟，在20秒的间隔。 2微升的KCN溶液（8微克/毫升）加入到每个孔中并记录另外2分钟的吸光度。计算KCN敏感的细胞色素c氧化复合物IV的活性。 </li></ol> 11。活性测定的F 0 F 1-ATP酶（ATP酶） <ol><li>准备F 0 F 1-ATP酶检测缓冲液（10mM的Tris-HCl，200 mM KCl中，2毫摩尔MgCl 2，pH 8.2）中。检测缓冲液中重悬线粒体颗粒，冻结线粒体样品的液体。三维氮气，慢慢地在冰上解冻样品。 </li><li>运行一式三份样品在48孔透明板。每个治疗组中，2的井将运行来测量总的ATP酶活性（275微升测定缓冲液，5微升线粒体悬浮液，和5微升95％乙醇中）和1，以及测量寡霉素不敏感的ATP酶活性（275微升测定缓冲液，5微升的线粒体悬浮液，和5微升的1毫克/毫升的寡霉素在95％乙醇溶液）。 </li><li>在31°C孵育10分钟恒定的颤抖。加入16微升的100 mM ATP溶液（pH 7.0），在31℃孵育5分钟，使用恒定的震动。 </li><li> 200μl的培养混合物转移到一个管中，50μl的3 M TCA。孵育样品在冰上10分钟，和自旋下来以10,000 xg离心5分钟，在4℃。使用上清颗粒，以确定磷酸盐和蛋白质含量分别。 </li><li>确定使用上清液中的磷酸盐含量无机磷酸盐的测定。准备加入0.88克硫酸亚铁4至10毫升3.75 MH 2 SO 4和调整到第90毫升，用H 2 O，100毫升的萨姆纳试剂按到该溶液中的FeSO 4在H 2 SO 4，加入10毫升的钼酸铵溶液（0.66克毫升H 2 O）。避光。 </li><li>负载96孔板中，用50μl每个磷酸标准（0 - 1,000μM的KH 2 PO 4）和50μl的每种上清液重复。每孔加入250μl的萨姆纳试剂的板在30°C孵育15分钟恒定的颤抖。 </li><li>记下在室温下在595 nm处的吸光度。 F 0 F 1-ATP酶活性，是由从ATP测量的寡霉素敏感的释放的 P i，正如我们前面所述。7,8计算总和寡霉素不敏感的F 0 F 1-ATP酶活性。计算寡敏感的ATP酶激活VITY，寡不敏感减去总ATP酶活性的活动。8,9 </li></ol> 12。细胞内ATP含量的测定<ol><li>将培养皿RPTC膜的冰，吸出培养基，用冰冷的PBS缓冲液洗两次单层。加入1 ml PBS中，各单层，刮去各单层的PBS中，并在Eppendorf管中收集的细胞悬浮液。附带的Eppendorf管中，在5秒的离心。 </li><li>确定在每个RPTC样品的ATP含量的荧光素酶的方法，使用的ATP生物发光检测试剂盒HS II（Roche）和制造商的协议。确定在每个样品中的蛋白质浓度，并计算每毫克蛋白。8的 ATP浓度</li></ol> 13。线粒体形态的可视化<ol><li>改变培养基。加入2μl100μM解决方案的的Mitotracker有红580每道菜（终浓度100纳米）和返回的菜的incubator为30分钟。 </li><li>检查活的单分子膜5在荧光显微镜下，用水浸物镜。 </li></ol> 14。细胞活力分析<ol><li>在开始测定之前，在24小时时，准备50mM的溶液中顺铂治疗，将作为阳性对照，细胞凋亡（膜联蛋白V-阳性细胞）的细胞。另外，准备500 mM的叔丁基过氧化氢 ​​的溶液中，将作为阳性对照胀亡（碘化丙啶（PI）阳性细胞）的细胞治疗。 </li><li>治疗2款2μl的50毫米顺铂和2款2μl的500毫米叔丁基过氧化氢 ​​。 1碟专用无染控制。 </li><li>在治疗与顺铂和TBHP的24小时后，制备的结合缓冲液（的10mM的Hepes，140 mM氯化钠，5mM的氯化钾，1毫摩尔的MgCl 2，1.8毫的CaCl 2）和碘化丙啶（PI）溶液（50微克/毫升）在结合缓冲液中。 </li><li>一次洗单分子膜有约束力的BUFF呃。添加1毫升的冰冷的缓冲液中，每个培养皿中，除了无染色和膜联蛋白V-阳性细胞的PI溶液和50μl。包括菜肴和冰上孵育15分钟，温柔的轨道晃动。 </li><li>洗净单层用结合缓冲液（10分钟），并加入1毫升的膜联蛋白V-FITC溶液每个培养皿中，除了没有污点和PI阳性细胞的结合缓冲液和2μl。盖菜，并在室温下孵育10分钟，温柔的轨道晃动。 </li><li>吸液的缓冲液和洗单层用1ml冰冷的结合缓冲液在冰上10分钟，温和的轨道摇动。重复洗涤步骤。 </li><li>吸结合缓冲液，加入500μl结合缓冲液，每道菜。轻轻地刮了的细胞用橡胶警察和转让的细胞，流式细胞仪可检测管。使细胞分散成单细胞悬浮液，通过移液样品向上和向下。分手的细胞团。 </li><li>立即FLO量化PI和Annexin V-FITC荧光瓦特仪使用PI和FITC在590和530 nm，激发波长为488 nm，发射。每个样本数为10,000个事件。 </li><li>将FITC荧光上的X轴方向和在Y-轴的流式细胞仪散点图数字PI荧光。被认为是细胞凋亡阳性细胞为Annexin V和负PI。被认为是细胞PI和Annexin V的负肿胀。10,11 </li></ol>

<ol>
	<li>Nowak, G., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+culture+conditions+stimulate+gluconeogenesis+in+primary+cultures+of+renal+proximal+tubule+cells.">Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells.</a> Am. J. Physiol. 268, C1053-C1061 (1995).</li><li>Nowak, G., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-ascorbic+acid+regulates+growth+and+metabolism+of+renal+cells:+improvements+in+cell+culture.">L-ascorbic acid regulates growth and metabolism of renal cells: improvements in cell culture.</a> Am. J. Physiol. 271, 2072-2080 (1996).</li><li>Ping, P., Takano, H., Zhang, J., Tang, X. L., Qiu, Y., Li, R. C., Banerjee, S., Dawn, B., Balafonova, Z., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoform-selective+activation+of+protein+kinase+C+by+nitric+oxide+in+the+heart+of+conscious+rabbits:+a+signaling+mechanism+for+both+nitric+oxide-induced+and+ischemia-induced+preconditioning.">Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning.</a> Circ. Res. 84, 587-604 (1999).</li><li>Wotton, D., Ways, D. K., Parker, P. J., Owen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+both+Raf+and+Ras+is+necessary+for+activation+of+transcription+of+the+human+T+cell+receptor+beta+gene+by+protein+kinase+C,+Ras+plays+multiple+roles.">Activity of both Raf and Ras is necessary for activation of transcription of the human T cell receptor beta gene by protein kinase C, Ras plays multiple roles.</a> J. Biol. Chem. 268, 17975-17982 (1993).</li><li>Nowak, G., Bakajsova, D., Samarel, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-&#949;+activation+induces+mitochondrial+dysfunction+and+fragmentation+in+renal+proximal+tubules.">Protein kinase C-&#949; activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules.</a> Am. J. Physiol. Renal Physiol. 301, F197-F208 (2011).</li><li>Nowak, G., Clifton, G. L., Godwin, M. L., Bakajsova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+ERK1/2+pathway+mediates+oxidant-induced+decreases+in+mitochondrial+function+in+renal+cells.">Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells.</a> Am. J. Physiol. Renal Physiol. 291, 840-855 (2006).</li><li>Shaik, Z. P., Fifer, E. K., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt+activation+improves+oxidative+phosphorylation+in+renal+proximal+tubular+cells+following+nephrotoxicant+injury.">Akt activation improves oxidative phosphorylation in renal proximal tubular cells following nephrotoxicant injury.</a> Am. J. Physiol. Renal Physiol. 294, F423-F432 (2008).</li><li>Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-&#945;+and+ERK1/2+mediate+mitochondrial+dysfunction,+decreases+in+active+Na++transport,+and+cisplatin-induced+apoptosis+in+renal+cells.">Protein kinase C-&#945; and ERK1/2 mediate mitochondrial dysfunction, decreases in active Na+ transport, and cisplatin-induced apoptosis in renal cells.</a> J. Biol. Chem. 277, 43377-43388 (2002).</li><li>Liu, X., Godwin, M. L., Nowak, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C-+a+inhibits+the+repair+of+oxidative+phosphorylation+after+S-(1,2-dichlorovinyl)-L-cysteine+injury+in+renal+cells.">Protein kinase C- a inhibits the repair of oxidative phosphorylation after S-(1,2-dichlorovinyl)-L-cysteine injury in renal cells.</a> Am. J. Physiol. Renal Physiol. 287, F64-F73 (2004).</li><li>Nowak, G., Price, P. M., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+a+functional+p21WAF1/CIP1+gene+accelerates+caspase-independent+apoptosis+induced+by+cisplatin+in+renal+cells.">Lack of a functional p21WAF1/CIP1 gene accelerates caspase-independent apoptosis induced by cisplatin in renal cells.</a> Am. J. Physiol. Renal Physiol. 285, F440-F450 (2003).</li><li>Cummings, B. S., Schnellmann, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cisplatin-induced+renal+cell+apoptosis:+caspase+3-dependent+and+-independent+pathways.">Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways.</a> J. Pharmacol. Exp. Ther. 302, 8-17 (2002).</li></ol>

没有利益冲突的声明。

这里介绍的方法允许个人同工酶的PKC的过度表达在肾小管上皮细胞的原代培养的。这种方法有几个优势：1。它允许在一个同质的人口调查监管机制的细胞（肾小管上皮细胞），各种损伤（缺血，缺氧，氧化应激），药物和nephrotoxicants肾脏内的主要目标。 2。 在体外模型的RPTC生长在原代培养条件的改善线粒体功能，在这个类似于肾近曲小管在体内的线粒体功能。1,2 3。线粒体?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>层流罩</td><td>热电公司</td><td> FORMA 1104 </td></tr><tr><td> 2毫升和15毫升DOUNCE组织研磨机</td><td> WHEATON </td><td> 989-24607，357544 </td></tr><tr><td> 85和235微米的尼龙网</td><td>小零件</td><td> CMN - 0085 - 10YD CMN - 0250 - 10YD </td></tr><tr><td> 50毫升无菌的离心管中</td><td> BIOLOGIX </td><td> BCT-P 50BS </td></tr><tr><td> 1.5毫升的微管</td><td>萨尔斯塔特</td><td> 72.690.001 </td></tr><tr><td> 35×10毫米的无菌培养皿</td><td>康宁</td><td> 430165 </td></tr><tr><td>儒安离心机</td><td>儒安</td><td> CR3 11175704转子：儒安儒安T40 </td></tr><tr&gt; <td>可调微离心</td><td> SIGMA </td><td>模型1 - 15 </td></tr><tr><td>生物氧气检测仪</td><td> YSI注册成立</td><td> YSI型号5300A </td></tr><tr><td>单室微型氧气系统</td><td> YSI注册成立</td><td> 5356S </td></tr><tr><td>氧探头</td><td> YSI注册成立</td><td> 5331A </td></tr><tr><td>循环浴</td><td> YSI注册成立</td><td> 5310 </td></tr><tr><td>氯化钾（KCl）和标准膜包</td><td> YSI注册成立</td><td> 5775 </td></tr><tr><td>磁力搅拌器</td><td> YSI注册成立</td><td> 5222 </td></tr><tr><td>平板式记录仪</td><td> KIPP＆Zonen公司</td><td> BD 11E </td></tr><tr><td> 48孔和96孔透明板</td><td>深圳科诗特</td><td> 3548，3679 </td></tr><tr><td>恒温ŕ </td><td> Ëppendorf </td><td> 5355 21919 </td></tr><tr><td> 2000年轨道摇床MAXQ </td><td> Thermo Scientific的</td><td> SHKA 2000 </td></tr><tr><td>光谱：FLUOR加（吸收/荧光/发光读者） </td><td> Tecan公司</td><td> F129005 </td></tr><tr><td>水套美国自动流CO 2自动孵化器</td><td> NUAIRE </td><td> NU 4850 </td></tr><tr><td>的12x75毫米聚苯乙烯文化流式细胞仪试管</td><td>费舍尔品牌</td><td> 14-961-20 </td></tr><tr><td> Axioskop 水浸物镜63X / 0,90 W </td><td>卡尔·蔡司</td><td> 114846 ACHROPLAN 44 00 67 </td></tr><tr><td> DMEM / F12 </td><td> Cellgro </td><td> 99 - 830 - PB </td></tr><tr><td> DMEM / F12火腿的</td><td>西格玛</td><td> ð2906 - 1L </td></tr><tr><td>去铁胺甲磺酸</td><td>住院RA </td><td> D110 </td></tr><tr><td> I型胶原酶</td><td>沃辛顿</td><td> 4196 </td></tr><tr><td>胰蛋白酶抑制剂</td><td>西格玛</td><td> Ţ6522 - 500MG </td></tr><tr><td>碘（JC-1）5,5&#39;，6,6&#39; - 四氯-1，1&#39;，3,3&#39; - tetraethylbenzimidazolylcarbocyanine </td><td> Invitrogen公司</td><td> T3168 </td></tr><tr><td> Mitotracker红</td><td> Invitrogen公司</td><td> M22425 </td></tr><tr><td> ATP生物发光检测试剂盒海关II </td><td>罗氏公司</td><td> 11 699 709 001 </td></tr><tr><td>膜联蛋白V - FITC解决方案</td><td>产品描述BioVision </td><td> 1001 - 200 </td></tr><tr><td>流式细胞仪</td><td> BD Biosciences公司</td><td> BD FACSCalibur </td></tr></tbody></table>

assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase c isozymes

同功酶的蛋白激酶C（PKC）家族参与了许多生理和病理过程。我们最近的数据表明，PKC调节线粒体的功能和细胞能量状态。许多报告表明激活PKC-αPKC-ε提高在缺血性心脏的线粒体功能和介导的心脏保护作用。相反，我们已经表明，PKC-α和PKC-ε参与在nephrotoxicant诱导的线粒体功能障碍和肾脏细胞中的细胞死亡。因此，本研究的目标是开发PKC同工酶可以选择性地激活或抑制，以确定其氧化磷酸化和细胞生存的调节作用，保持积极的线粒体功能的肾细胞的体外模型。培养更好的条件，导致线粒体线粒体呼吸和活动的原代培养肾小管上皮细胞（RPTC）线粒体酶类似于那些在RPTC 体内 。由于传统的转染技术（脂质体转染，电穿孔）在原代培养中是低效的，对线粒体功能产生不利影响，PKC-ε突变cDNA分别传递到RPTC通过腺病毒载体。这种做法的结果转染培养RPTC超过90％。 在这里，我们提出了评估的作用，PKC-ε的方法：1。调节线粒体形态和ATP合成，和2与相关联的功能。 RPTC在小学文化的生存期。由过度的组成性激活PKC-ε突变激活PKC-ε。 PKC-ε抑制过度的无效突变体的PKC-ε。线粒体功能的评估是通过检查呼吸，呼吸链的完整性，活动，呼吸复合物和F 0 F 1-ATP酶，ATP生成率和ATP含量。呼吸是ssessed的毛地黄皂苷-透性RPTC状态3（最大呼吸中存在的多余的基板和ADP）和非耦合呼吸。呼吸链的完整性进行评估，通过测量所有4个配合物中分离线粒体呼吸链的活动。氧化磷酸化的能力的评价通过测定线粒体膜电位，ATP生成率，和F 0 F 1-ATP酶的活性。的RPTC的能源状况进行评估，确定细胞内ATP含量。在活细胞中的线粒体形态可视化使用Mitotracker有红580，专门蓄积在线粒体内的荧光染料，和活的单层，在荧光显微镜下检查。 RPTC可行性评估采用膜联蛋白V /碘化丙啶染色后流式细胞仪测定细胞凋亡和胀亡。 这些方法允许在个别的PKC同功酶的选择性激活/禁止，以评估它们的作用，在细胞功能中的各种生理和病理条件，可以在体外再现。

图1表明，腺病毒交付的cDNA编码的组成型活性（caPKC-ε）和非活动状态（dnPKC-ε）的突变体的PKC-ε的查询结果的显着增加的蛋白水平RPTC和线粒体中的PKC-ε。用cDNA携带caPKC-ε载体感染的细胞过表达PKC-ε的磷酸化形式（有源）而感染的细胞的cDNA编码dnPKC-ε过度表达PKC-ε是不活动的（未磷酸化的）（ 图1）。存在的活性PKC-ε降低线粒体呼吸RPTC无论通电线粒体而无效的PKC-ε并无效?...

Watch this Scientific Journal Video about Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes at JoVE.com

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

活化的蛋白激酶C（PKC）的同工酶与呼吸，氧化磷酸化有关的线粒体功能和对细胞活力的影响进行说明。该方法适应腺病毒技术，选择性过度表达PKC同工酶在原代培养和各种检测，以确定线粒体功能和能量状态的细胞。

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

assessment-mitochondrial-functions-cell-viability-renal-cells

Research

JoVE Journal

Biology

18.1K 次观看.  University of Arkansas for Medical Sciences. 活化的蛋白激酶C（PKC）的同工酶与呼吸，氧化磷酸化有关的线粒体功能和对细胞活力的影响进行说明。该方法适应腺病毒技术，选择性过度表达PKC同工酶在原代培养和各种检测，以确定线粒体功能和能量状态的细胞。

视频: Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

计数和确定培养细胞的可行性

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

科研

生物学

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

激活细胞凋亡的细胞色素的细胞质显微注射 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Assessment of GFP Expression and Viability Using the Tali Image-Based Cytometer

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide different information. Flow cytometry gives quantitative multi-parametric information about physical characteristics and staining or expression, but doesn't allow for visualization. Stand-alone fluorescence microscopy provides visual data, but doesn't allow for straightforward quantitative measurements1.
Image-based cytometry bridges the gap between these two methods, enabling the quick visualization and simultaneous quantitative analysis of thousands of cells in heterogeneous populations2. Here, we present a method for performing cell viability and green fluorescent protein (GFP) expression assays using the Tali Image-Based Cytometer3. The Tali instrument is a 3-channel (bright field, green fluorescence, red fluorescence) benchtop assay platform that offers several advantages over flow cytometry and fluorescence microscopy. The Tali cytometer is less expensive, takes up less bench space, requires less maintenance, and the work flow has been simplified so that the operation and analysis is much simpler and quicker. The Tali cytometer is capable of performing a range of suspension cell-based assays, including GFP and red fluorescent protein (RFP) expression, apoptosis4-6 and cell viability analysis with propidium iodide (PI)7-11.
Here, we demonstrate the use of the Tali instrument in performing a cell viability assay in cells expressing GFP. GFP-transduced cells are stained using the Tali Viability Kit - Dead Cell Red. The cells are then pipetted into a Tali Cellular Analysis Slide and loaded into the cytometer. Bright field, red fluorescence and green fluorescence images are captured and analyzed using assay specific algorithms. Histograms are then generated to display cell size, PI fluorescence intensity, and GFP fluorescence intensity. These parameters can then be thresholded to home in on a specific cell population.
A side-by side comparison of the Tali Image-Based Cytometer and traditional flow cytometry demonstrates that the two methods provide comparable data regarding cell viability and protein expression. However, the Tali instrument provides additional visual information about the cell population that cannot be obtained using a flow cytometer.

This protocol describes how to perform cell viability and fluorescence expression assays using the Tali Image-Based Cytometer.

GFP的表达和使用的大里基于图像流式细胞仪的可行性评估

Single-cell and population information are commonly obtained either by flow cytometry or fluorescence microscopy. However, these two methods provide ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

在活细胞中蛋白质聚集的4D成像

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

活性检测在培养细胞

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2&#8242;-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches available to study mitochondrial localization and activity of a cell cycle kinase. These are advanced, yet easy to follow methods that will be beneficial to many cell biology researchers.

Here, we outline how to study mitochondrial localization of a (cell cycle) kinase, and how to determine its sub-mitochondrial location as well as potential mitochondrial substrates/targets. Forced expression of proteins into the mitochondria provides a useful tool for studying the functional consequences of mitochondrial localization of a protein of interest.

实验方法来研究线粒体定位和核细胞周期激酶CDK1功能

Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

线粒体钙和线粒体膜的同时测量通过荧光显微镜活细胞的潜在

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase (ERK) Signaling or Mitochondrial Events in Target Cells

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation can simultaneously activate multiple cellular molecular signaling pathways. In this protocol, we show that through coupling femtosecond laser into a confocal microscope, cells can be stimulated precisely by the tightly-focused laser. Some molecular processes that can be simultaneously observed are subsequently activated. We present detailed protocols of the photostimulation to activate extracellular signal regulated kinase (ERK) signaling pathway in Hela cells. Mitochondrial flashes of reactive oxygen species (ROS) and other mitochondrial events can be also stimulated if focusing the femtosecond laser pulse on a certain mitochondrial tubular structure. This protocol includes pretreating cells before photostimulation, delivering the photostimulation by a femtosecond laser flash onto the target, and observing/identifying molecular changes afterwards. This protocol represents an all-optical tool for related biological researches.

Photostimulation by Femtosecond Laser Activates Extracellular-signal-regulated Kinase ERK Signaling or Mitochondrial Events in Target Cells

Tightly-focused femtosecond laser can deliver precise stimulation to cells by being coupled into a confocal microscopy enabling the real-time observation and photostimulation. The photostimulation can activate cell molecular events including ERK signaling pathway and mitochondrial flashes of reactive oxygen species.

飞秒激光的光刺激激活目标细胞中的细胞外信号调节激酶(ERK)信号或线粒体事件

Direct control of cellular defined molecular events is important to life science. Recently, studies have demonstrated that femtosecond laser stimulation ...

评估肾细胞​​蛋白激酶C同工酶，线粒体功能及细胞活力的

评估肾细胞蛋白激酶C同工酶，线粒体功能及细胞活力的