我们感谢英国医学研究委员会 (U105115237) 对 RRK 的核心资助。

1. 细胞和材料的制备
<ol><li>在 SM 琼脂板上培养细胞与细菌如产气克雷伯杆菌, 或在营养培养基如 HL5 如所述23。 注意: 生长在 macropinocytosis 上的突变体在细菌中有缺陷有助于防止次生突变的积累, 从而增加 macropinocytosis 的发生率。将通道编号保持在4以下, 然后从库存中镀出电池以获得最佳效果。突变体应在隔离后尽快储存为冷冻储存。</li>
	<li>在 sm 培养基上培养细胞, 生长K 产气以融合在 sm 介质中。添加 200–300xg ul 的细菌到 SM 琼脂板和传播。采取无菌循环的细胞 (当从另一个细菌板块转移), 并在板块的一个边缘扩散。孵育22摄氏度, 长达一周。</li>
	<li>将四甲基罗丹明异硫氰酸酯 (TRITC) 葡聚糖溶于水中50毫克/毫升, 过滤器使用0.22 微米过滤器, 整除数1毫升, 贮存在-20 摄氏度。整除数可以无限期地保留下来。 注: 155 kDa 是与此方法使用的典型尺寸, 因为它可以廉价购买散装, 但较小的 dextrans 测量 macropinocytosis 就像盘基柄24一样有效。如果需要不同的显影, 则不同的非具有易点燃 dextrans (如层叠蓝色或 Alexa-647) 是可选的。</li>
</ol>2. 将定性测量转化为定量 (可选)
<ol><li>
使用悬浮液中的细胞进行液体摄取测定。
	<ol>
<li>颗粒 axenically 生长细胞在 300 x g 3 分钟, 丢弃上清和并用重悬在营养培养基 1x107细胞/毫升。添加 TRITC-葡聚糖到0.5 毫克/毫升和动摇 180 rpm, 22 °c。</li>
		<li>稀释0.8 毫升样品到0.7 毫升冰冷的 kk2在 0, 30, 60, 90, 120 分钟洗涤一次在1.5 毫升冰冷的 kk2缓冲23在一个台式离心机, 并用重悬到1毫升在同一缓冲和存放在冰上。 注: 立即洗涤, 或一旦所有样品已收集产生等效结果。</li>
	</ol>
</li>
	<li>
确定细胞内部的流体体积。
	<ol>
<li>设置 fluorimeter 测量荧光使用544毫微米的激发波长和发射波长574毫微米与10毫微米狭缝。建立了 TRITC-葡聚糖的稀释系列, 并用它测定了葡聚糖给定体积的荧光量。创建校准曲线。</li>
		<li>用0.9 毫升细胞悬浮液测量2.1 节细胞内化的荧光。使用校准曲线24计算每个细胞内化的流体体积。</li>
	</ol>
</li>
	<li>稀释其余的细胞从每个时间点分别成0.5 毫升冰冷 KK2。过滤通过70µm 细胞过滤器到5毫升聚苯乙烯流式细胞术管。设置流式细胞仪, 使每个时间点的单元格彼此不同, 并记录其荧光 (见3.4 和3.5 节)。</li>
	<li>使用上面的设置测量流式细胞仪中校准珠子的荧光, 并记录它。 注: 这是所有未来荧光测量流式细胞仪的参考: 在使用再次运行的珠子和调整设置, 使他们有相同的荧光。这将给出一个非常狭窄的定义峰值。在荧光峰周围放置一个门可以使它变得更容易。</li>
	<li>重复三次并绘制2.2 和2.3 节中获得的细胞荧光数据。绘制最佳拟合线, 并利用该方程将流式细胞仪中的定性数据转换为定量单位。</li>
</ol>3. 测量流体摄取量
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58434/58434fig1.jpg"> 图 1: macropinocytosis 的高通量测量示意图。(A)在与K. 产气细菌 (黄色)盘基柄的 SM 板上生长。收获细胞从喂养前面 (橙色), 避免已经开发的细胞 (绿色), 成25毫升的 KK2缓冲。旋涡到离解, 颗粒3分钟离心在 300 x g, 然后洗3次在50毫升的 KK2缓冲, 每次丢弃上清。并用重悬到 1 x 105细胞/毫升在 HL5 生长介质中, 并添加50µL 到三井每个样品的平底96井板。孵育在22°c 24 小时(B)稀释 TRITC 葡聚糖到1毫克/毫升在 HL5 生长培养基从50毫克/毫升的股票溶液。添加50µL 到每个样品 (不包括0分钟摄取控制井), 并孵化22°c 1 小时后, 添加葡聚糖到0分钟摄取控制。(C)立即醒酒媒体, 在纸巾上拍上盘子, 去除多余的培养基, 浸泡在冰冷的 KK2缓冲液中, 填满水井冲洗。醒酒的缓冲和拍干再次。添加100µL 5 毫米叠氮化钠溶解在 KK2MC, 以分离细胞。采取流式细胞仪测量内化荧光。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58434/58434fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol><li>
建立一个平底的96井组织培养板 (图 1A)。使用细菌生长的细胞, 并将其转移到 HL5 培养基 (含有100µg/毫升的双氢链霉素, 100 µg/毫升氨苄西林和50µg/毫升卡那霉素) 24 小时之前的化验;这使得 macropinocytosis 可以从24细菌生长的细胞中的低水平上调。或者, 直接从无菌培养中稀释细胞, 在化验前孵化24小时, 以减少由于细胞从不同密度稀释而产生的误差。<ol>
<li>在50毫升离心管中从喂养前收获细胞到25毫升的 KK2 。分离细胞由涡流和颗粒在 300 x g 3 分钟.丢弃上清液, 并用重悬颗粒, 在 300 x克3 分钟内清洗3次, 在50毫升的 KK2, 每次丢弃上清液去除细菌。</li>
		<li>确定细胞密度 (使用 hemacytometer 或其他细胞计数系统), 稀释成含有抗生素的 HL5 1 x 105细胞/毫升。吸管 50 ul 入每个井, 使用三井为每个情况。孵育22°c 为24小时. 记得设置0分钟的摄取控制。 注: 可替代培养基,如有罪、VL6。</li>
	</ol>
</li>
	<li>
用 TRITC-葡聚糖加载细胞 (图 1B)。
	<ol>
<li>稀释葡聚糖到1毫克/毫升的培养基使用 (这可以增加到2.5–5毫克/毫升当评估细胞非常低摄取)。添加 50 ul 的每一个井 (给最终浓度0.5 毫克/毫升 TRITC 葡聚糖), 并返回到22°c 1 h, 因为这允许明显的葡聚糖积累, 但胞吐作用的葡聚糖尚未开始。 注: 中继吸管允许此步骤更快地完成, 减少错误。</li>
	</ol>
</li>
	<li>
为流式细胞术准备细胞 (图 1C)。
	<ol>
<li>在洗涤之前, 增加 50 ul 含葡聚糖的媒介到0分钟摄取控制。</li>
		<li>醒酒的培养基和轻拍干燥的组织。将盘子浸入冰冷的 KK2, 然后醒酒。 注意: 有些粘附缺陷的菌株可能会在这一步骤中分离出来,例如塔林 (塔拉-talB-)25的两个同系物的挖空。当使用单元格线时, 如果需要的话, 请小心连接不良和吸出介质。</li>
		<li>添加 100 ul 5 毫米叠氮化钠溶解在 kk2MC (kk2 + 2 毫米 MgSO4和100微米 CaCl2)。 注意: 叠氮化钠极具毒性。使用粉尘面罩和安全眼镜时, 与粉末。总是戴手套和实验室大衣。避免加热, 不要与酸混合。 注意: 细胞迅速分离, 胞吐作用被阻止24。显微镜可以用来检查这个。</li>
	</ol>
</li>
	<li>
流式细胞仪测量流体摄取量
	<ol>
<li>如果计划将相对值转换为绝对值, 则使用珠子标准化流式细胞仪设置 (见第2节)。或者, 使用以葡聚糖加载的单元格, 如在2节中, 以确保正确设置正散射和侧向散射以隔离单元格 (图 2A), 确保机器未被阻止, (图 2B) 并将参数调整为测量内化荧光 (图 2C)。</li>
		<li>附加高通量采样系统并添加板。建立一个测量荧光的协议 (为 TRITC 葡聚糖使用黄绿色激光激发和测量在582毫微米渠道或相似) 65 ul 的细胞悬浮并且跑。</li>
	</ol>
</li>
	<li>
流式细胞术结果分析
	<ol>
<li>使用正向散射和侧散射的单元格上的门 (图 2A)。使用 "统计" 选项计算单元格的中值荧光。使用一个示例设置这些参数, 并应用于所有示例。 注意: 使用不同的突变体或抑制剂时, 正向散射/侧向散射剖面可以改变样品。</li>
		<li>确定每个条件下三井的平均值, 减去0分钟的摄取控制。使用2节中确定的等式或正常化到该控件, 可以转换为量化值。</li>
	</ol>
</li>
</ol>4. 执行流体吸收时间-课程
<ol><li>将单元格设置为3节, 每个时间点和应变/条件有三个井。</li>
	<li>按顺序将葡聚糖标记培养基添加到不同的井中。典型的时间路线测量流体吸收在 0, 30, 60, 90, 120 和180分钟. 在180分钟的时间点井中添加葡聚糖培养基, 随后60分钟后将其加入到井中, 用于120分钟时间点等(图 3A)。</li>
	<li>在0分钟, 添加 50 ul 的葡聚糖培养基和立即醒酒和洗盘子, 如第3.3 节。分析如第3.5 节所述。</li>
</ol>5. 剂量响应曲线
<ol><li>按照3节所述设置单元格, 每个应变和条件有三个井。</li>
	<li>将所感兴趣的化合物稀释成含有1毫克/毫升葡聚糖的培养基, 使化合物的最大最终浓度加倍。用与第一个相同比例的葡聚糖制备第二管含培养基。漩涡混合。</li>
	<li>创建一个稀释系列的兴趣化合物在 200 ul 葡聚糖含培养基每样 (图 4A)。漩涡混合。在1小时内将培养基的 50 ul 添加到每个样品中。</li>
	<li>洗涤和分析, 如第3.3 节所述。</li>
</ol>6. 吞噬和膜吸收
<ol><li>要测量膜的摄取量, 请按照3节的说明准备细胞。添加 50 ul, 其中含有20微米 FM 1-43, 最后浓度为10微米。加载后, 冲洗和准备细胞的流式细胞术, 如3.3 节。测量在 585 nm 通道或类似, 使用蓝色激光激发。 注: 由于膜的贩运速度比液相快, 10 分钟是一个较合适的时间点使用比1小时26。替代染料, 荧光只有当加入到膜可以代替。</li>
	<li>
使用荧光标记的珠子或细菌来测量吞噬功能.荧光酵母不能使用, 因为它们是不可分离的盘基柄的向前和侧面分散24。按照第3节中的说明设置单元格, 并按描述的27进行分析。<ol>
<li>测量珠子的吞噬功能添加黄绿色标签珠, 如第3.2 节所述, 最终浓度为 5 x 107珠/毫升 (1.75 和2微米) 或 1 x 108珠/毫升 (1 和1.5 微米), 在洗涤前和准备流式细胞术之前, 如3.3 节。测量在 525 nm 通道或类似, 使用蓝色激光激发。 注意: 高浓度会导致细胞脱离。</li>
		<li>为了测量细菌的吞噬功能, 添加德州红色标签大肠杆菌 bioparticles, 如3.2 至 1 x 108细菌/毫升在洗涤前1小时, 并准备流式细胞术, 如3.3 节所述。测量在 610 nm 通道或类似, 使用黄绿色激光激发。</li>
	</ol>
</li>
</ol>

<ol>
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A., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum+mutants+with+temperature-sensitive+defects+in+endocytosis.">Dictyostelium discoideum mutants with temperature-sensitive defects in endocytosis.</a> Journal of Cell Biology. 127, 387-399 (1994).</li><li>Basu, S., Fey, P., Jimenez-Morales, D., Dodson, R. J., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dictyBase+2015:+Expanding+data+and+annotations+in+a+new+software+environment.">dictyBase 2015: Expanding data and annotations in a new software environment.</a> Genesis. 53 (8), 523-534 (2015).</li><li>Williams, T. D., Kay, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiological+regulation+of+macropinocytosis+during+Dictyostelium+growth+and+development.">The physiological regulation of macropinocytosis during Dictyostelium growth and development.</a> Journal of Cell Science. 131 (6), (2018).</li><li>Tsujioka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+functions+of+the+two+talin+homologues+in+Dictyostelium.">Overlapping functions of the two talin homologues in Dictyostelium.</a> Eukaryotic Cell. 7 (5), 906-916 (2008).</li><li>Aguado-Velasco, C., Bretscher, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulation+of+the+plasma+membrane+in+Dictyostelium.">Circulation of the plasma membrane in Dictyostelium.</a> Molecular Biology of the Cell. 10, 4419-4427 (1999).</li><li>Sattler, N., Monroy, R., Soldati, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+phagocytosis+and+phagosome+maturation.">Quantitative analysis of phagocytosis and phagosome maturation.</a> Methods in Molecular Biology. 983, 383-402 (2013).</li><li>Wilkins, A., Chubb, J., Insall, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+Dictyostelium+RasGEF+is+required+for+normal+endocytosis,+cell+motility+and+multicellular+development.">A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development.</a> Current Biology. 10, 1427-1437 (2000).</li><li>Thomason, P. A., King, J. S., Insall, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mroh1,+a+lysosomal+regulator+localized+by+WASH-generated+actin.">Mroh1, a lysosomal regulator localized by WASH-generated actin.</a> Journal of Cell Science. 130 (10), 1785-1795 (2017).</li><li>van Duijn, B., Vogelzang, S. A., Ypey, D. L., van der Molen, L. G., van Haastert, P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+chemotaxis+in+Dictyostelium+discoideum+cells+with+a+depolarized+plasma+membrane+potential.">Normal chemotaxis in Dictyostelium discoideum cells with a depolarized plasma membrane potential.</a> Journal of Cell Science. 95, 177-183 (1990).</li><li>Novak, K. D., Peterson, M. D., Reedy, M. C., Titus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+myosin+I+double+mutants+exhibit+conditional+defects+in+pinocytosis.">Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis.</a> Journal of Cell Biology. 131, 1205-1221 (1995).</li><li>Mercer, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccinia+virus+uses+macropinocytosis+and+apoptotic+mimicry+to+enter+host+cells.">Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells.</a> Science. 320 (5875), 531-535 (2008).</li><li>Paschke, P., 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=Rapid+and+efficient+genetic+engineering+of+both+wild+type+and+axenic+strains+of+Dictyostelium+discoideum.">Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum.</a> PLoS One. 13 (5), e0196809 (2018).</li><li>Bloomfield, G., Tanaka, Y., Skelton, J., Ivens, A., Kay, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+duplications+in+the+genomes+of+laboratory+stocks+of+Dictyostelium+discoideum.">Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum.</a> Genome Biology. 9 (4), R75 (2008).</li><li>Fey, P., Dodson, R. J., Basu, S., Chisholm, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+stop+shop+for+everything+Dictyostelium:+dictyBase+and+the+Dicty+Stock+Center+in+2012.">One stop shop for everything Dictyostelium: dictyBase and the Dicty Stock Center in 2012.</a> Methods in Molecular Biology. 983, 59-92 (2013).</li></ol>

作者没有披露的利益冲突。

而其他评估流体吸收的方法是低吞吐量,就地洗涤细胞和使用叠氮化钠分离细胞是这种方法的关键步骤, 允许高通量测量 macropinocytosis, 膜吸收, 或盘基柄吞噬。当细胞被连接到一个表面, 而培养基不是, 它们可以被附加, 而在它们周围的介质首先被抛出, 然后通过沉浸在缓冲和再次抛出。叠氮化钠, 耗尽细胞 ATP 和 depolarizes 膜30, 然后用于分离细胞, 也防止胞吐作用不影响细胞活力24。
当使用流式细胞仪测量 macropinocytosis 的盘基柄给出非常准确的测量液体摄取非常迅速, 以确定为什么一个特定的应变或条件改变了流体摄取, 进一步调查使用显微镜是必需的24。还应指出的是, 以前公布的结果在某些情况下表明, 在表面上生长的突变株 (如本例), 或在震动悬浮 (如标准议定书)31中, 液体吸收的差异。使用这种方法可能意味着, 在罕见的情况下, 明显的液体摄取缺陷被遗漏。另外, 在测量吞噬功能时, 只能使用低浓度的微粒。这种技术可以确定的吞噬功能的最大速率远远低于实际的最大值, 尽管仍然可以测量菌株与条件之间的吞噬功能的相关差异24。为了确定吞噬功能的最大速率, 必须通过备选协议27来测量震动悬浮中的吸收量。有吞噬珠子的细胞增加了侧向散射, 因此在设置流式室仪时应相应地改正。
流式细胞术可用于测量哺乳动物细胞中的液体摄取量32, 然而, 其他吞通路比盘基柄所见的更高比例的液相吸收是一个值得关注的问题。此外, 细胞通常被分离使用胰蛋白酶在37°c, 允许进一步吞进展的内聚糖酐。冰冷叠氮化钠不会导致巨噬细胞从表面分离 (威廉斯, 未发表的观察), 使得这种技术不适用于哺乳动物细胞而没有进一步的优化。
高通量测量 macropinocytosis 有可能被用来快速和廉价地筛查抑制剂, 基因突变或基因击倒在盘基柄细胞的影响。变种人应该总是和他们的直系父母相比。如果读者没有事先偏爱盘基柄菌株, 非无菌菌株, 如 DdB 或 NC4 比无菌更 "野生型", 可以有效地操作无菌菌株33。否则, Ax2 菌株是基因组重复度最少34的无菌菌株, 而 Ax4 的许多菌株塔林一个挖空, 如果可能的话, 应该避免23。大多数以前发布的菌株可以从 Dicty 股票中心订购35。
这种技术允许比以前更大的调查可能性的影响, 不同的条件, 抑制剂和突变对 macropinocytosis 的盘基柄。

macropinocytosis 的大规模非特异流体吸收在几个生物学环境中是重要的1, 包括免疫细胞抗原取样2, 病原体进入宿主细胞3, 癌细胞增殖4和传播朊病毒疾病5。在哺乳动物和盘基柄细胞中, 肌动蛋白6,7, PI (34, 5) P38,9,10 (虽然脂质的确切性质不同的两个11), 激活Ras12,13和活化 Rac14,15是重要的有效的流体吸收 macropinocytosis, 虽然仍然有许多悬而未决的问题, 关于如何 macropinocytic 补丁是形成, 组织和最终内化。发现更多的蛋白质对 macropinocytosis 重要, 并随后确定它们如何在不同的生物学环境中是重要的, 将给予更全面的理解 macropinocytosis, 并可能允许发展有针对性的治疗范围的条件。
盘基柄是研究 macropinocytosis 的理想模型系统。标准实验室菌株本构 macropinocytosis 的高水平意味着, 由于 macropinocytosis6, 流体摄取量超过90%。这使得 macropinocytosis 仅通过确定流体摄取量来测量, 不同于哺乳动物细胞, 因为 macropinocytosis 的液体摄取比例要低得多。macropinocytosis 是如此清楚地定义和容易地可视化12在这个系统相似地提供独特的好处为调查 macropinosome 的核心保守的组分在其他系统可能有多个调控信号16,17。
用于测量哺乳动物细胞 macropinocytosis 的标准技术涉及在短时间内用葡聚糖脉冲固定细胞, 然后用显微术来确定由葡聚糖阳性囊泡18所占据的细胞的面积。然而这项技术不考虑 macropinosomes 收缩的可能性, 当进入细胞, 在盘基柄19报告了, 并且只考虑了细胞的单平面, 意味容量内部化尚不清楚。一种替代技术, 计算在给定时间内内化的 macropinosomes 的数量, 有相同的缺点20。使用盘基柄避免了这些问题;然而, 现有的测量盘基柄液体摄取的技术是相对劳力密集型的, 使用大量的细胞和葡聚糖21。细胞在高密度的荧光葡聚糖和样品去除在不同的时间点, 以确定内化荧光使用 fluorimeter。这种方法制备的细胞可以通过流式细胞仪来分析, 以获得单细胞, 而不是人口级的分辨率22, 尽管这仍然是低吞吐量的。
在这里, 用荧光葡聚糖作为标签来测量内化流体的标准技术适应了96井板格式, 用流式细胞仪分析了使用高通量取样 (高温超导) 附件的样品。细胞被喂食非具有易点燃荧光葡聚糖的预先确定的时间长度, 浸泡在冰冷的缓冲和分离使用5毫米叠氮化钠, 这也停止胞吐作用。然后用流式细胞仪对各井的细胞进行分析。该方法的目的是克服上述方法的局限性, 并允许同时比较的液体吸收大量的菌株/条件, 同时使用较少的资源和减少劳动所涉及的。

<table>
	<tbody>
		<tr>
			<td>LSR_II flow cytometer</td>
			<td>BD Biosciences</td>
			<td>-</td>
			<td>Other Flow cytometers can also do this role, e.g. the LSRFortessa by BD</td>
		</tr>
		<tr>
			<td>TRITC-dextran (155 kDa)</td>
			<td>Sigma-Aldrich</td>
			<td>T1287</td>
			<td>Other non-quenchable dextrans, and other sizes are also fine</td>
		</tr>
		<tr>
			<td>HL5 medium</td>
			<td>Formedium</td>
			<td>HLGCFG</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plate</td>
			<td>Corning</td>
			<td>3596</td>
			<td>Any flat-bottom tissue culture treated 96-well plate will work</td>
		</tr>
		<tr>
			<td>Dihydrostreptomycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1517</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium</td>
			<td>Formdium</td>
			<td>AMP50</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin monosulfate</td>
			<td>Sigma-Aldrich</td>
			<td>60615</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>VWR</td>
			<td>103694M</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate hydrate</td>
			<td>VWR</td>
			<td>25169.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>VWR</td>
			<td>1.02382.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phopshate</td>
			<td>VWR</td>
			<td>1.04877.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-potassium hydrogen phosphate</td>
			<td>VWR</td>
			<td>1.05104.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorimeter</td>
			<td>Perkin-Elmer</td>
			<td>LS 50 B</td>
			<td></td>
		</tr>
		<tr>
			<td>FM1-43</td>
			<td>Thermofisher</td>
			<td>T35356</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresbrite YG-carboxy microspheres 1.00 &#181;m</td>
			<td>Polysciences</td>
			<td>15702-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresbrite YG-carboxy microspheres 1.50 &#181;m</td>
			<td>Polysciences</td>
			<td>09719-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresbrite YG-carboxy microspheres 1.75 &#181;m</td>
			<td>Polysciences</td>
			<td>17687-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoresbrite YG-carboxy microspheres 2.00 &#181;m</td>
			<td>Polysciences</td>
			<td>09847-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red E. coli bioparticles</td>
			<td>Thermofisher</td>
			<td>E2863</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow-set fluorospheres</td>
			<td>Beckman Coulter</td>
			<td>6607007</td>
			<td>Calibration Beads</td>
		</tr>
		<tr>
			<td>SM agar</td>
			<td>Formedium</td>
			<td>SMACFG</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#181;m syringe filter</td>
			<td>Elkay Laboratory Products</td>
			<td>E25-PS22-50S</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>302188</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-bottom polystyrene tubes</td>
			<td>Corning</td>
			<td>352058</td>
			<td>Use a tube that will fit onto your flow cytometer.</td>
		</tr>
		<tr>
			<td>70 &#181;m cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tube</td>
			<td>Sarstedt</td>
			<td>62.547.004</td>
			<td></td>
		</tr>
		<tr>
			<td>Repeating pipette</td>
			<td>Eppendorf</td>
			<td>M4-SK</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL repeating pipette tips</td>
			<td>Eppendorf</td>
			<td>30089650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>LY294002</td>
			<td>Cayman Chemical Company</td>
			<td>70920</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404-2MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput measurement of dictyostelium discoideum macropinocytosis by flow cytometry

macropinocytosis 对某些癌细胞的增殖、抗原取样、宿主细胞侵袭和神经退行性疾病的传播都具有重要的意义。柄虫盘基柄的常用实验室菌株在营养培养基中生长时具有极高的流体吸收率, 其中超过90% 是由于 macropinocytosis。此外, 哺乳动物 macropinocytosis 的许多已知核心成分也存在, 这使得它成为研究 macropinocytosis 的优秀模型系统。在这里, 用荧光葡聚糖作为标签来测量内化流体的标准技术适应了96井板格式, 用流式细胞仪分析了使用高通量取样 (高温超导) 附件的样品。
细胞被喂食非具有易点燃荧光葡聚糖的预先确定的时间长度, 浸泡在冰冷的缓冲和分离使用5毫米叠氮化钠, 这也停止胞吐作用。然后用流式细胞仪对各井的细胞进行分析。该方法还可用于测量荧光珠或细菌的膜吸收和吞噬功能。
这一方法的设计, 以允许测量流体吸收的盘基柄在高通量, 劳动力和资源有效的方式。它允许同时比较多个菌株 (如基因的挖空突变体) 和条件 (如不同培养基中的细胞或不同浓度的抑制剂) 平行和简化时间课程。

一旦该技术已经完成, 细胞被装上葡聚糖并准备进行分析 (图 1), 确保流式细胞仪不会被阻止, 并调整正散射/侧散射剖面, 看起来像图 2A所示的单元格。如果计算机被阻止, 它看起来会更像图 2B所示, 并且必须在继续之前取消阻塞。确保参数显示控制无菌细胞有较高的内化葡聚糖荧光在较长的时间点和低内化荧光在较短的 (图 2C)。
当寻找变种人之间的差异, 很可能会有三表型。变种人可能有正常的体液摄取, 他们可能有部分缺陷, 或液体摄取可以完全废除。图 2D显示了一个正常流体摄取的应变, 在这种情况下, 标准实验室应变 Ax2, 一个突变体的液体摄取量减少了 50% (Ax2 rasG-14) 和一个与被废除的液体摄取 (Ax3 gefB-28)。获得平均中流体摄取量 (3.5 节), 并使用它来计算内部流体的体积 (如图 3B所示), 或者将数据与控件 (如图 4B 和4C) 进行比较。
当执行流体吸收时间路线, 如图 3A, 内化荧光应增加 60–90 min, 之后葡聚糖开始 exocytosed 和一个高原达到 (图 3B)。使用60分钟作为标准时间点时, 比较 macropinocytosis 在不同的突变体/条件, 因此, 使一个良好的信号, 并没有失去信号, 由于胞吐作用。胞吐作用严重受阻的突变体可能需要较长的时间才能到达高原29。
当治疗细胞的抑制剂是有效的抗 macropinocytosis 在盘基柄(如图 4A), 葡聚糖内化1小时将下降到几乎没有在较高的抑制剂浓度在大多数情况下(图 4B)。一些抑制剂可能不是100% 有效的, 但是,例如nocodazole 只抑制多达50% 的液体摄取 macropinocytosis 时, 增加敏锐 (图 4C)。如果抑制剂是无效的, 细胞将内部化类似数量的葡聚糖作为控制, 并减少荧光将不会看到。这种技术允许大量不同的抑制剂和抑制剂浓度被筛选, 以影响对流体吸收的 macropinocytosis 非常迅速, 减少了花费的时间优化抑制剂处理。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58434/58434fig2.jpg"> 图 2: 设置流式细胞仪和代表性数据。(A)应设置细胞的正散射 (FSC) 和侧向散射 (SSC) 剖面, 以便能够很容易地区分细胞。显示 Ax2 单元格的外观的示例。(B)如果流式细胞仪被阻断, 如本例中所示, 单元格的侧向散射非常低。激光不会正确地激发显影, 机器在继续之前应该被解除阻塞。在计算机被阻止时获得的任何数据都应被丢弃。(C)应设置荧光, 使0分钟摄取样本具有低荧光, 当细胞在荧光介质中孵化时间较长时会增加, 如从威廉姆斯 & 凯 201824中抽取的例子所示。(D)以正常 macropinocytosis (Ax2、绿色)、减少 macropinocytosis (Ax2 rasG、HM172614、橙色) 和废除 macropinocytosis (Ax3 gefB) 为1小时的 TRITC 葡聚糖培养的细胞的例子,HM177628, 蓝色)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58434/58434fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58434/58434fig3.jpg"> 图 3: 在96井板中执行流体吸收时间-课程。(A)应按顺序将葡聚糖添加到每组样品中, 完成时间相同。然后用流式细胞仪对井进行冲洗, 分离和测量内化荧光。在这里显示添加葡聚糖的例子次数。(B)在96井板中进行的 Ax2 细胞的吸液时间过程。取自威廉姆斯 & 凯 201824, 误差条显示三独立实验的标准误差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58434/58434fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58434/58434fig4.jpg"> 图 4: 流体摄取剂量响应曲线。(A)增加兴趣的化合物, 在这种情况下, PI3K 抑制剂 LY294002, HL5 含有1毫克/毫升 TRITC 葡聚糖在双倍的预期最终最大浓度。与 HL5 生长培养基 + 1 毫克/毫升葡聚糖混合在不同比例单独的车辆, 以产生稀释系列200µL 培养基的每一个条件。在洗涤和测量内化荧光之前, 将井作为正常的1小时。(B)用 LY294002-containing 培养基从A中孵化的 Ax2 细胞的吸液剂量响应曲线。改编自威廉姆斯 & 凯 201824。液体摄取被规范化为未经处理的控制。误差条显示三独立实验的标准误差。(C)与 nocodazole 一起孵化的 Ax2 细胞的吸液剂量响应曲线。改编自威廉姆斯 & 凯 201824。液体摄取被规范化为未经处理的控制。误差条显示三独立实验的标准误差。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58434/58434fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry at JoVE.com

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

Macropinocytosis, 大规模的非特定液体摄取, 是重要的许多领域的临床生物学包括免疫学, 感染, 癌症和神经退行性疾病。在这里, 现有的技术已经适应了允许高通量, 单细胞分辨率测量 macropinocytosis 在 macropinocytosis 模型生物盘基柄柄使用流式细胞仪。

流式细胞仪检测盘基柄柄Macropinocytosis 的高通量

Large-scale non-specific fluid uptake by macropinocytosis is important for the proliferation of certain cancer cells, antigen sampling, host cell invasion ...

high-throughput-measurement-dictyostelium-discoideum-macropinocytosis

Nanyang Technological University

Research

JoVE Journal

Biology

High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

7.7K 次观看.  MRC-Laboratory of Molecular Biology, Cambridge, UK. Macropinocytosis, 大规模的非特定液体摄取, 是重要的许多领域的临床生物学包括免疫学, 感染, 癌症和神经退行性疾病。在这里, 现有的技术已经适应了允许高通量, 单细胞分辨率测量 macropinocytosis 在 macropinocytosis 模型生物盘基柄柄使用流式细胞仪。

视频: High-throughput Measurement of Dictyostelium discoideum Macropinocytosis by Flow Cytometry

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

IP - FCM:免疫沉淀，流式细胞仪检测

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

科研

生物学

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Flow Cytometry Purification of Mouse Meiotic Cells

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the unique differentiation programs that are manifest during meiosis. Two principal methods have been developed to purify, to varying degrees, various meiotic fractions from both adult and immature animals: elutriation or Staput (sedimentation) using BSA and/or percoll gradients. Both of these methods rely on cell size and density to separate meiotic cells1-5. Overall, except for few cell populations6, these protocols fail to yield sufficient purity of the numerous meiotic cell populations that are necessary for detailed molecular analyses. Moreover, with such methods usually one type of meiotic cells can be purified at a given time, which adds an extra level of complexity regarding the reproducibility and homogeneity when comparing meiotic cell samples.
Here, we describe a refined method that allows one to easily visualize, identify, and purify meiotic cells, from germ cells to round spermatids, using FACS combined with Hoechst 33342 staining7,8. This method provides an overall snapshot of the entire meiotic process and allows one to highly purify viable cells from most stage of meiosis. These purified cells can then be analyzed in detail for molecular changes that accompany progression through meiosis, for example changes in gene expression9,10and the dynamics of nucleosome occupancy at hotspots of meiotic recombination11.

An efficient method to obtain highly purified viable meiotic fractions from mouse testis is described, which combines a refined cell dissociation protocol with fluorescent activated cell sorting (FACS). This method takes advantage of differences in the DNA content and nuclear density of discrete meiotic fractions.

流动小鼠减数分裂细胞的流式细胞仪净化

The heterogeneous nature of cell types in the testis and the absence of meiotic cell culture models have been significant hurdles to the study of the ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

高通量测量和环境样品中的有机磷分类

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

优化细胞分裂监测细胞示踪染料的染色和扩散建模方法

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

外层膜蛋白的分离线粒体的流式细胞仪免疫检测

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers1-4. This process is dependent on the release of cyclic adenosine monophosphate (cAMP)5. cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors6,7. A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions8,9. This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)10. Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities11,12. Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade10. The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.

Investigating the Function of Coronin A in the Early Starvation Response of Dictyostelium discoideum by Aggregation Assays

The social amoeba Dictyostelium discoideum undergoes a developmental transition into a multicellular organism when starved. The evolutionary conserved protein coronin A plays a crucial role in the initiation of development. Using aggregation assays as our main method, we aim to elucidate the role of coronin A in early development.

调查Coronin A在早期饥饿反应中的作用<em&gt;粘菌</em&gt;通过聚集测定

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular ...

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium discoideum revealed that a complex signal transduction network of parallel pathways amplifies the response to chemoattractants, and leads to biased actin polymerization and protrusion of a pseudopod in the direction of a gradient. In contrast, molecular mechanisms driving other types of directed migration, for example, due to exposure to shear flow or electric fields, are not known. Many regulators of chemotaxis exhibit localization at the leading or lagging edge of a migrating cell, as well as show transient changes in localization or activation following global stimulation with a chemoattractant. To understand the molecular mechanisms of other types of directed migration we developed a method that allows examination of cellular response to acute mechanical stimulation based on brief (2 - 5 s) exposure to shear flow. This stimulation can be delivered in a channel while imaging cells expressing fluorescently-labeled biosensors to examine individual cell behavior. Additionally, cell population can be stimulated in a plate, lysed, and immunoblotted using antibodies that recognize active versions of proteins of interest. By combining both assays, one can examine a wide array of molecules activated by changes in subcellular localization and/or phosphorylation. Using this method we determined that acute mechanical stimulation triggers activation of the chemotactic signal transduction and actin cytoskeleton networks. The ability to examine cellular responses to acute mechanical stimulation is important for understanding the initiating events necessary for shear flow-induced motility. This approach also provides a tool for studying the chemotactic signal transduction network without the confounding influence of the chemoattractant receptor.

Assessment of Dictyostelium discoideum Response to Acute Mechanical Stimulation

Here we describe methods for assessing cellular response to acute mechanical stimulation. In the microscopy-based assay, we examine localization of fluorescently-labeled biosensors following brief stimulation with shear flow. We also test activation of various proteins of interest in response to acute mechanical stimulation biochemically.

对急性机械刺激的网柄反应的评估

Chemotaxis, or migration up a gradient of a chemoattractant, is the best understood mode of directed migration. Studies using social amoeba Dictyostelium ...