作者感谢中西部大学的研究和赞助项目的支持。

1. 制备各种哺乳动物细胞系厌氧培养培养基
<ol><li>
为缺氧细胞培养制作完整的 PS-74656 培养基25。
	<ol>
<li>在50毫升蒸馏去离子水中溶解17.25 克亚硝酸根, 使无菌亚硝酸盐库存溶液 (5 米; 100x), 然后过滤消毒。</li>
		<li>添加0.5 毫升的亚硝酸盐库存溶液每50毫升低葡萄糖 DMEM (1 克/升葡萄糖) 与110毫克/升的 l-谷氨酰胺, 584 毫克/升的丙酮酸钠, 10% 胎牛血清 (血清; 热灭活)。 注: 使用血清浓度大于10% 是有毒的。值得注意的是, 一条癌细胞线对亚硝酸盐 (MDA-MB-231, 乳腺癌细胞系) 的培养基耐受性低, 因此在没有亚硝酸盐的情况下生长。此外, 虽然 PS-74656 培养基支持所有经测试的细胞系的生长 (n = 9), 某些细胞系 (例如, 细胞) 表现出较高的细胞浓度和较高的生存能力 PS-74656 高葡萄糖 (4.5 克/升) DMEM25。</li>
		<li>在无菌50毫升圆锥离心管中放置20毫升培养基。不要拧紧盖子;它应该是松散的。</li>
		<li>将管子放在真空钟罐中, 用近似45°角将其脱气, 使介质表面积最大化。</li>
		<li>在室温下使用真空泵或其等效的真空。保持真空, 直到气泡不再被观察 (24–48 h)。</li>
		<li>把管子从罐子里取下, 然后立即关上瓶盖, 封好管子, 尽量减少空气进入介质中的引入。</li>
		<li>把管子放在商业厌氧室里。</li>
		<li>松开管帽, 允许管内的空气与燃烧室中的厌氧气体混合物平衡至少24小时。 注: 介质应留在会议厅内, 直至用完为止。为加快工艺速度, 用缺氧气体混合液将管至少3x 冲洗, 用无菌转移吸管填充缺氧气, 由 H2、CO2和 N2 组成.
</li>
		<li>将所有管 (10–15分钟) 放置在厌氧腔内, 如上文所述, 在密封管和放置在室内。使用前, 使用1.5–2毫升转移吸管或吸管提示, 冲洗它与厌氧气体混合物, 已冲洗的缺氧气体至少3x。</li>
		<li>在厌氧室中, 将介质中的100µL 移除无菌脱气微离心管, 并在腔内使用氧气探针对溶解氧进行测试。 注: 氧电极在使用前按制造商的协议进行校准。正确脱气和平衡厌氧气体混合介质的读数为0。 注意: 如果读数超过 0.3 ppm 的氧气, 继续在厌氧室孵化介质, 因为有更多的时间平衡培养基是必需的。</li>
	</ol>
</li>
</ol>2. 各种哺乳动物细胞系的缺氧培养
<ol><li>
指定的天-1
	<ol>
<li>生长细胞 (37 °c, 5% CO2) 在 T150 瓶到90% 汇合为缺氧和常氧文化控制提供足够数量的细胞为三 24-井板材 (80% 汇合)。 注意: 对于这项研究, 使用了 HeLa 229 和维罗细胞。该议定书也经过测试, 并证明成功的增长和维护七其他细胞线25。</li>
		<li>从烧瓶中取出培养基, 吸入10毫升的 HBSS。</li>
		<li>用5毫升的胰蛋白酶取代培养基。搅动烧瓶, 直到细胞从塑料中分离出来。吸入大部分的胰蛋白酶, 然后轻敲烧瓶以去除黏附细胞。添加10–15毫升的高葡萄糖 DMEM (4.5 克/升葡萄糖, 110 毫克 l-谷氨酰胺, 10% 血清, 50 µg/毫升的庆大霉素), 以灭活胰蛋白酶。</li>
		<li>Triturate 细胞使用25毫升的吸管, 直到所有细胞团簇被破坏。</li>
		<li>用标准 hemocytometer 和台盼蓝染料排除法或自动当量测定细胞数并确定其可行性。</li>
		<li>悬浮在高葡萄糖 DMEM 的细胞, 如上所述, 细胞密度为 2.24 x 105细胞/毫升。</li>
		<li>将1毫升的细胞悬浮液添加到24井组织培养板 (2.24 x 105细胞/井; 80% 汇流) 井中。种子1板为缺氧培养和另一个控制常氧文化。</li>
		<li>孵化细胞在37°c 在常氧条件 (5% CO2) 为 24 h。 注: 这些细胞可以在各种大小的板材中电镀。然而, 种子细胞为缺氧生长的烧瓶, 有风险, 引入氧气进入系统, 除非采取谨慎, 以彻底冲洗出大气气体。</li>
	</ol>
</li>
	<li>
指定的天0
	<ol>
<li>常氧孵化24小时后, 将板块用于缺氧生长进入厌氧室 (商业厌氧室;图 1)。</li>
		<li>立即将培养基改为无抗生素的脱气 PS-74656 培养基, 并将其驯化为24小时至厌氧室。将盘子放在一个带有湿巾的容器里, 并松散地关闭以保持湿气。 注: 常氧生长板的处理方式相同, 例外的是所有的治疗都是在大气条件下进行的。</li>
	</ol>
</li>
	<li>
指定的天1
	<ol>
<li>1天的厌氧孵化开始后24小时的孵化在商业厌氧室, 其中包含标准的厌氧气体混合物10% 小时2, 10% CO2, 80% N2。</li>
		<li>在不同的时间段孵化细胞。</li>
		<li>监测厌氧介质的颜色变化随着时间的推移。 注意: 从红橙到洋红色的颜色转换信号的时间改变媒介。这可能需要长达2周的时间才能发生。从红色橙色到黄色的培养基中的颜色变化表明存在细菌污染。</li>
		<li>当介质从红色变为洋红时, 通过吸移去除 runagate 细胞。</li>
		<li>将吸气用过的介质放在脱气和厌氧气体平衡离心管中的 runagate 细胞中, 关闭管, 同时确保密封安全, 然后将其离心 (326 x克; 室温为10分钟)。立即用1毫升新的脱气 PS-74656 培养基替换井中的吸气介质。</li>
		<li>在打开它之前, 将离心管放在厌氧室中的颗粒细胞。吸入所用的培养基, 并用新鲜的脱气 PS-74656 培养基替换离心管中的总容积1毫升。</li>
		<li>将细胞从管中返回到24井组织培养板中的井中。</li>
	</ol>
</li>
</ol>3. 厌氧培养细胞的显微观察对表型细胞分化的评价
<ol><li>
在厌氧室中, 将盘子放在一个密封盒中, 用厌氧气体混合物冲洗, 然后关闭盒子。 注: 盒内必须装有湿纸巾, 以提供防潮, 防止板材干燥。对于显微镜, 三明治袋的工作最好, 因为他们是薄的。<ol>
<li>观察显微镜下的细胞, 完全密封袋并将其从腔内取出。</li>
		<li>仔细拉伸塑料袋在盘子上, 检查细胞使用倒置相对比显微镜与相机附件, 并采取显微照片在各种放大。</li>
		<li>将密封袋中的盘子退回到房间, 并按照步骤2.3.3 中的描述继续孵化。</li>
		<li>要单独分析处于悬浮中的 runagate 细胞, 请按照步骤2.3.5 中概述的步骤进行操作。 注: 这些细胞可以用来进行有氧测试, 也可以被送回厌氧室, 用于测试这个特定的人群。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

这种方法代表第一次哺乳动物细胞, 在没有氧气的情况下长时间培养。根据目前的观察,通过非发酵途径的缺氧生长能力似乎是普遍的哺乳动物细胞28, 其中厌氧生长导致表型发散。这是观察到所有细胞系测试。以厌氧耕种, 细胞的增加的比例变得四舍五入, 开发了悬浮象人口, 并且能复制。次级细胞类型仍然附着在基板上;然而, 细胞形态学恢复到主轴 (树突状) 形状。这些发?...

细胞从固体肿瘤, 干细胞的利基, 和那些衬里的粘膜表面存在的环境中, 有氧水平降低, 包括缺氧1,2,3,4。在正常的生理状态下, 氧合不同于缺氧的缺氧 (完全没有氧气)5,6。在二十世纪七十年代代初, 发现大气氧对哺乳动物细胞复制产生不利影响, 并且在耗尽的氧气条件下可以优化体外细胞生长。里氏等。7表明, 1–3% 氧水平 (缺氧) 提高电镀效率与大气氧 (20%)。在缺氧培养条件下, 人类双倍细胞寿命也在8。
在体内, 缺氧条件发生时, 氧储存耗尽 (例如, 在激烈的运动期间), 其中 ATP 生产从骨骼肌切换到有氧呼吸到发酵 (厌氧呼吸) 与乳酸的末端产品9。病理上, 在癌性肿瘤中, 肿瘤肿块的内部是缺氧的, 因为血管化10的不良。有限灌注对肿瘤内部的影响是独立的, 由强制厌氧1所殖民的肿瘤内部进行验证。机械化, 在缺氧的肿瘤细胞生存被认为完全依赖于缺氧诱导因子1α基因 (HIF1) 的表达, 这是最初自发反应缺氧4,11,12. HIF1在缺氧条件下诱发的热休克蛋白结合HIF1启动子和 upregulate 基因转录12。这些热休克蛋白被认为有助于诱导的各种表型在肿瘤缺氧微环境。这些表型表现了细胞膜葡萄糖转运体的增加的表示和糖酵解的率 (华宝效应)13。其结果是从线粒体氧化磷酸化转变为乳酸发酵。
缺氧生存也可以利用替代葡萄糖来支持生存现象14,15。最好的研究哺乳动物的例子是鼹鼠, 它可以生存近20分钟没有氧气通过果糖驱动的酵发酵途径14。另一种适应发生在某些鱼类 (如鲤鱼 (鲫鱼 ), 它可以存活明显较长的时间, 使用糖酵解与乙醇作为终端副产品)15。在这两种情况下, 发酵驱动新陈代谢使生存在缺乏氧气。目前的缺氧生存假说是, 只要HIF1在缺氧期间激活, 线粒体呼吸, 不需要氧气, 发生在厌氧条件下16。此外, 据推测, 使用发酵途径的缺氧/缺氧生存, 提高肿瘤生存, 因为细胞避免氧化应激, 可能证明是有害的细胞生存17。最近的一项研究表明, 在心肌细胞中, 缺氧会降低肿瘤细胞17的氧化应激, 这一假说得到了证实。
到目前为止, 缺氧哺乳动物细胞生存的发酵通路的本质是在文献中根深蒂固的, 这在很大程度上是由于无法在完全没有氧气的情况下培养哺乳动物细胞3天以上。然而, 在细菌中, 厌氧生存的一种替代糖酵解。在某些细菌中, 氮或硫酸盐 (在其他化合物中) 可以作为细胞色素氧化酶系统的终端电子受体, 在没有氧18的情况下。虽然细菌和真核进化发生在平行的, 因为从最后一个普遍的共同祖先, 估计线粒体是提供能量的细胞在154.2万年前的氧合的海洋19.由于研究人员已经表明, 孤立的线粒体可以在没有氧气的情况下产生 ATP, 以亚硝酸盐作为终端电子受体, 假设细胞在没有氧气的情况下可以在3天的时间内正常工作。20,21,22,23. 本研究所述的方法对许多细胞系的厌氧哺乳动物细胞生长有效用。

<table border="0" cellpadding="0" cellspacing="0" style="width:709px;" width="709">
	<tbody>
		<tr height="147">
			<td height="147" style="height:147px;width:255px;">Whitney A35 anaerobic chamber</td>
			<td style="width:149px;">Don Whitley Scientific&#160;</td>
			<td style="width:139px;">Microbiology International</td>
			<td style="width:167px;">The ability to remove the front of the chamber makes it easy to put instruments inside without concern about getting them through the portals.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">CO2 incubator</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:139px;">3531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue Culture Hood</td>
			<td style="width:149px;">Labconco</td>
			<td style="width:139px;">DO55735</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">pipet aid</td>
			<td style="width:149px;">Drummond</td>
			<td style="width:139px;">4-000-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sterile transfer pipets</td>
			<td style="width:149px;">Santa Cruz</td>
			<td style="width:139px;">sc-358867</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">50cc sterile conical centrifuge tubes</td>
			<td style="width:149px;">DOT</td>
			<td style="width:139px;">451-PG</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">vaccum jar</td>
			<td style="width:149px;">Nalgene 111410862889</td>
			<td style="width:139px;">BTA-Mall 5311-0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM high glucose (4.5 g/L)</td>
			<td style="width:149px;">CellGro</td>
			<td style="width:139px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DMEM low glucose (1 g/L)</td>
			<td style="width:149px;">CellGro</td>
			<td style="width:139px;">10-014-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">FBS</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">1500-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">HBSS</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">trypsin</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">25-052-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">gentamicin</td>
			<td style="width:149px;">Gibco</td>
			<td style="width:139px;">15750-060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">sterile pipets</td>
			<td style="width:149px;">CellTreat</td>
			<td style="width:139px;">229210B, 229205B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue culture flask (T75 or T150)</td>
			<td style="width:149px;">Santa Cruz</td>
			<td>sc-200263, sc-200264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">24 well tissue culture treated dishes</td>
			<td style="width:149px;">DOT</td>
			<td style="width:139px;">667124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">glad ziplock sandwich bags</td>
			<td style="width:149px;">Ziploc</td>
			<td style="width:139px;">Costco</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">inverted phase microscope (10, 20 40x objectives with camera mont)</td>
			<td style="width:149px;">Nikon Eclipse</td>
			<td style="width:139px;">TS100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">trypan blue</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:139px;">T10282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">hemocytometer</td>
			<td style="width:149px;">Invitrogen</td>
			<td style="width:139px;">C10283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Countess Automated Cell Counter</td>
			<td style="width:149px;">Life Technologies</td>
			<td style="width:139px;">AMQAF1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Rainin microtiter pipets</td>
			<td style="width:149px;">Mettler Toledo</td>
			<td style="width:139px;">Rainin Classic Pipette PR-100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">microtiter tips</td>
			<td style="width:149px;">Santa Cruz Biotechnology</td>
			<td style="width:139px;">sc-213233 &#38; sc-201717&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">test tube rack (50cc tubes)</td>
			<td style="width:149px;">The Lab Depot</td>
			<td>HS29050A</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:255px;">sodium nitrite</td>
			<td style="width:149px;">Sigma</td>
			<td style="width:139px;">https://www.sigmaaldrich.com/catalog/product/sigald/237213?lang=en&#38;region=US</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">clear boxes with lids</td>
			<td style="width:149px;">Rosti Mepal</td>
			<td style="width:139px;">Rubber maid</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">paper towels</td>
			<td style="width:149px;">In House</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">cell scraper</td>
			<td style="width:149px;">CellTreat</td>
			<td style="width:139px;">229310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PBS</td>
			<td style="width:149px;">In house prepared</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">70% Ethanol</td>
			<td style="width:149px;">Fisher Scientific</td>
			<td style="width:139px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">microcentrifuge tubes sterile</td>
			<td style="width:149px;">Santa Cruz</td>
			<td style="width:139px;">sc-200273</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">biohazard bags with holder (desktop)</td>
			<td style="width:149px;">Heathrow Scientific</td>
			<td style="width:139px;">HS10320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nitrile Gloves</td>
			<td style="width:149px;">VWR</td>
			<td style="width:139px;">89428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">oxygen electrode</td>
			<td style="width:149px;">eDAQ</td>
			<td style="width:139px;">EPO354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH meter</td>
			<td style="width:149px;">Jenway</td>
			<td style="width:139px;">3510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pH paper/ pHydrion</td>
			<td style="width:149px;">Sigma Aldich</td>
			<td>Z111813</td>
			<td></td>
		</tr>
	</tbody>
</table>

anaerobic growth and maintenance of mammalian cell lines

大多数黏膜表面连同中点在肿瘤和干细胞龛位是身体的地理区域缺氧。先前的研究表明, 在常氧 (5% CO2的空气) 或缺氧 (低氧水平) 条件下, 缺氧孵化 (缺乏自由氧) 的孵化导致生存能力有限 (2–3天)。开发了一种新的方法, 使缺氧细胞培养 (至少17天; 测试的最大时间)。这一方法最关键的方面是确保不将氧气引入系统。这是由介质的脱气, 并通过冲洗管, 盘子, 烧瓶和吸管与厌氧气体混合物 (H2, CO2, N2) 后, 允许材料平衡缺氧 (非氧气) 环境在使用之前。在获取显微照片时必须进行额外的护理, 以确保所获得的显微照片不包含工件。在缺乏氧气的情况下, 细胞形态明显改变。所有厌氧细胞都有两种截然不同的 morphotypes。在缺乏氧气的情况下, 培养和维持哺乳动物细胞的能力可以应用于细胞生理学、polymicrobial 相互作用的分析以及新型癌症药物开发的生物合成途径的表征。

这个协议的力量在于它支持的长寿和多细胞系的生长, 并承认有深刻的变化和分化的细胞形态学25。这项研究最关键的因素是在严格的 anaerobiosis 下细胞的转移和维持。这就需要组织一个厌氧室来最大化该协议 (图 1), 并保证在密封的环境中保存用于显微镜或其他测试的细胞。到目前为止, 缺氧生存的范围是15–17天为不朽的癌细胞系和...

Watch this Scientific Journal Video about Anaerobic Growth and Maintenance of Mammalian Cell Lines at JoVE.com

Anaerobic Growth and Maintenance of Mammalian Cell Lines

哺乳动物细胞系的厌氧生长与维持

在这里, 我们描述了一种新的方法, 使厌氧长期培养已建立的细胞系。测试的最大生存时间是17天。该方法适用于细胞毒性药物的检测和 anoxically 复制细胞的生理学研究。

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

anaerobic-growth-and-maintenance-of-mammalian-cell-lines

Research

JoVE Journal

Biology

10.0K 次观看.  Midwestern University. 在这里, 我们描述了一种新的方法, 使厌氧长期培养已建立的细胞系。测试的最大生存时间是17天。该方法适用于细胞毒性药物的检测和 anoxically 复制细胞的生理学研究。

视频: Anaerobic Growth and Maintenance of Mammalian Cell Lines

Passaging HuES Human Embryonic Stem Cell-lines with Trypsin.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts of cell culture, and tend to acquire karyotypic abnormalities with high population doublings.  Proper passaging is essential for maintaining a healthy, undifferentiated, karyotypically normal HuES human embryonic stem cell culture.  First, an expanding culture is washed in PBS to remove residual media and cell debris, then cells are overlaid with a minimal volume of warm 0.05% Trypsin-EDTA.  Trypsin is left on the cells for up to five minutes, then cells are gently dislodged with a 2mL serological pipette.  The cell suspension is collected and mixed with a large volume of HuES media, then cells are collected by gentle centrifugation.  The inactivated trypsin media mixture is removed, and cells resuspended in pre-warmed HuES media.  An appropriate split ratio is calculated (generally 1:10 to 1:20), and cells re-plated onto a 1-2 day old plate containing a monolayer of irradiated mouse embryonic fibroblast feeder cells.  The newly seeded HuES culture plate is left undisturbed for 48 hrs, then media is changed every day thereafter.  It is important not to trpsinize down to a single cell suspension, as this increases the risk of introducing karyotypic abnormalities.

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin. Brought to you by JoVE.

人类胚胎干细胞传代的色调线与胰蛋白酶

In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.  Human embryonic stem cells are artifacts ...

科研

生物学

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

在哺乳动物细胞中细胞周期位置分析

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

测定哺乳动物细胞计数，细胞的大小和使用末席ž迷你自动细胞计数细胞的健康

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

中血红素的合成水平在哺乳动物细胞中测量

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...