作者实验室的工作得到了德国福雄格梅因沙夫特（http://www.dfg.de） 的支持;CO 1139/1-1）、化学工业基金会（http://www.vci.de/fonds）和哥廷根分子生物学中心（GZMB）。作者要感谢约尔格·斯图尔克对手稿的有益评论和批判性解读。

1. 准备阿加板块、文化媒体、冷冻库和前文化
<ol><li>准备生长介质和所需的试剂（参见材料和试剂表）。</li>
	<li>条纹B.亚提利菌株（例如BP40 （罗克格+ 古德布CR amyE：:P古德布- yfp）和 BP52 （罗克格+ 古德布+ 艾米： :P古德布- cfp）分别表示一个和两个活跃的 Gdhs）2，将用于 SP 中等 agar 板的竞争实验， 以获得单一菌落。在 37 °C 处连夜孵化板。</li>
	<li>以单个菌落和接种无菌培养管，含有4毫升LB液体介质。在 28 °C 和 220 rpm 的夜间生长细菌。</li>
	<li>在 28 °C 的夜间培养中生长，以避免细胞裂解或孢子。</li>
	<li>使用标准光谱仪测量波长为 600 nm （OD600）的培养物的光学密度。</li>
	<li>如果培养物已达到 OD600 的 2.0，将 0.75 毫升培养物与 1.5 毫升反应管中的无菌 50% 甘油溶液混合 0.75 毫升。最终的OD600 应该是1.0，以获得含有约108 个细胞/毫升的低温素。</li>
	<li>将管子存放在-80°C。</li>
	<li>对标有 cfp 和 yfp 编码氟化基因的每个菌株进行三种预培养。接种预培养（含有4毫升LB液体介质的无菌培养管）与1个μl细胞从-80°C低温库存。在 28 °C 和 220 rpm 的夜间孵化文化。</li>
</ol>2. 细菌共生、样品收集和样品储存
<ol><li>新鲜准备 100 毫升 C-Glc 和 CE-Glc 最小介质（参见试剂和材料表），并将每个介质的 20 毫升转移到无菌 100 毫升摇动烧瓶中。</li>
	<li>以一夜之间生长的 0.1 毫升预栽培为例，用 1.5 毫升的 1.5 毫升小面包中 0.9 毫升 LB 介质稀释它们，并确定 OD600。</li>
	<li>对于竞争实验，采取1.0-1.5 之间具有类似OD 600的不同菌株的预培养。</li>
	<li>为了获得混合细胞群，稀释具有适当OD600 的预培养细胞到20毫升C-Glc和CE-Glc中以100毫升摇瓶补充的0.05的OD600。 对于比赛实验，两个菌株应以1：1的比例混合。</li>
	<li>从烧瓶中取出 10 毫升样品，将其转移到 15 毫升塑料管中，在标准桌面离心机中通过离心机在 4，000 x g 中通过离心机采集细胞 10 分钟，然后丢弃超自然物。</li>
	<li>将细胞以0.5毫升新鲜的LB介质中补充，并将细胞转移到无菌的1.5毫升反应杯中。加入 0.5 毫升 50% 无菌甘油，通过严格的涡流混合悬浮，并将样品存放在 -80 °C 冰柜中，直到进一步治疗。</li>
	<li>在 37 °C 和 220 rpm 时，将留在摇动烧瓶中的细胞孵化长达 24 小时。将摇瓶放在黑暗中，以防止荧光片的照片漂白。</li>
	<li>在 7 小时和 24 小时的生长后，进一步采集 0.1 毫升的样本。测量并注意样品的1：10 稀释的 OD 600（C-Glc 或 CE-Glc 最小介质）。</li>
	<li>从每个培养物中取出适量的细胞，使低温素具有 OD600 的 1.0。将样品储存在-80°C，直到进一步治疗。</li>
</ol>3. 定量分析的样品处理、电镀和孵化
<ol><li>收集完所有样品后，解冻冷冻库，并在0.9%的盐溶液（见试剂和材料表）中稀释细胞，至多10-3。</li>
	<li>SP中等胶板上10-3 稀释的板0.1毫升，使用无菌玻璃移液器分配细胞。</li>
	<li>在黑暗中以 37 °C 的夜间孵化板，直到出现单个菌落。</li>
</ol>4. 通过立体荧光显微镜计算幸存者进行定量分析
<ol><li>用黑色笔将阿加板底部分成四部分，以便更好地定位，同时在立体荧光显微镜下计算幸存者。</li>
	<li>将板倒置在显微镜下，并使用冷光源将菌落聚焦。</li>
	<li>一旦菌落聚焦，切换到相应的过滤器集 CFP，以可视化 cfp标记菌株的幸存细胞。用笔给殖民地贴上标签并注明数字，来计算这种菌株的幸存者。</li>
	<li>用乙醇去除标签，切换到YFP过滤器集，以可视化 yfp标记菌株的幸存细胞。再次计算幸存者，用笔给殖民地贴上标签并注明数字。</li>
</ol>5. 半量子分析的样品处理和显微镜
<ol><li>对于说明性数字，在 SP agar 板上从步骤 3.1 中发现 10-4稀释（约 100 个细胞）中的 10 μl（见图 3）。</li>
	<li>在黑暗中以 37 °C 的夜间孵化板，直到出现单个菌落。</li>
	<li>将无盖的培养皿放在显微镜下，并使用冷光源将点聚焦。</li>
	<li>拍摄斑点的照片，以图示数字。选择适当的曝光时间，用冷光源拍摄殖民地的照片。</li>
	<li>无需移动板，请更改为 CFP 滤镜集，调整曝光时间并拍照。使用 YFP 过滤器设置进行相同的处理，并将图片保存以供进一步分析。</li>
</ol>6. 数据分析
<ol><li>使用 Excel 等软件进行定量数据分析。根据计算的殖民地，计算黄殖民地和蓝殖民地占整个殖民地数的百分比，该殖民地的百分比设定为100%。</li>
	<li>使用计算的数字创建堆叠条图（参见 图 4 和图 5）。使用 Adobe 照片商店等图像处理程序构建从不同地点拍摄的照片的合并图片。或者，可从 <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> 下载的免费软件 ImageJ 可用于图像处理。</li>
	<li>打开使用 CFP 过滤器集和 YFP 过滤器集拍摄的单个位置的图片。优化对比度和亮度，以减少来自媒体的任何背景荧光。</li>
	<li>转到其中一张图片并选择所有图片。复制图片并粘贴到另一张图片上。</li>
	<li>要么使用功能"颜色闪避"合并图片，要么使用通道选项卡覆盖荧光图片。来自不同媒体和不同时间点的殖民地合并图片代表了液体文化中不同菌株的生长。</li>
</ol>7. 具体提示：染料开关实验和同源菌株的培养，标有 cfp 和 yfp
B.亚提利中两个氟编码基因中任何一个的表达可能会影响细菌的生长速度。因此，建议进行以下实验，以排除在培养过程中从细胞群中消除一个竞争对手菌株只是由于氟磷的负面影响：
<ol><li>重复整个实验并共生反标记的菌株（例如，较早 的 cfp标记菌株现在标记为 yfp 基因， 反之亦然）。虽然相反，但获得的结果应与先前的观察结果相媲美，即一种菌株比另一株具有选择性生长优势。</li>
	<li>重复整个实验，并共同培育标有yfp和cfp的同源菌株 （例如BP40（rocG+ gudBCR amyE：:P古德布- yfp）和 BP41 （罗克格+ 古德布CR 艾米： :P古德布- cfp））。通过跟踪细胞种群的组成，估计两种氟磷中的任何一种对细菌的适应力的负面影响。</li>
</ol>

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作者宣称他们没有相互竞争的经济利益。

已经开发了几种方法来分析细菌16的竞争性健康。在许多情况下，细菌被标记为不同的抗生素耐药性盒17。与我们的方法类似，用抗生素耐药盒标记细胞可以评估在规定的生长条件下共生过程中细菌的竞争性健康。此外，这种方法可用于确定17号染色体上特定轨迹中彼此不同的细胞的竞争性适应性。然而，使用抗生素耐药盒来监测竞技体能存在一些缺点。由于耐药基因的表?...

土壤细菌通常具有灵活的调控网络和广泛的代谢能力。这两个特征使细胞能够调整其催化物和合成代谢途径，以与研究员和其他微生物竞争营养物质，这些营养物质在给定的生态利基1中可用。然而，如果细菌不能适应其环境，其他机制可能决定一个物种的生存。事实上，由于许多细菌迅速增殖，种群可以达到高细胞密度亚种群，可能自发地积累了有益的突变，为细胞提供了选择性的生长优势，从而增加了细胞的适应能力。此外，突变热点和压力引起的适应性突变可以促进一种不适应细菌2，3的进化。因此，在连续选择下突变的积累和生长是巨大的微生物多样性的起源，即使在同一属4，5。与自然界一样，由于在选择下不断培养，细菌基因组的形成也会在实验室中发生。Gram阳性细菌B.亚提利的驯化就是例证，该细菌在全世界用于基础研究和工业。在20世纪40年代，B.亚提利斯接受了DNA损伤X射线治疗，随后在特定的生长条件下进行了培养。细菌在驯化过程中积累的突变导致许多生长特征的丧失，即B.亚提利实验室菌株168丧失了形成复杂菌落的能力7，8。
如今，对于研究最好的大肠杆菌和B.亚提利菌模型，各种强大的工具可用于基因操纵其基因组，以解决特定的科学问题。有时，兴趣基因的失活会导致严重的生长缺陷，然后在标准生长介质9上清晰可见。相比之下，导致生长缺陷微弱，从而只轻微影响菌株适应的突变往往被忽视。然而，在这两种情况下，长期潜伏和传递的突变菌株几代人通常会导致抑制突变体的积累，已经恢复了表型的父菌株2，9。抑制突变体的特征和突变的识别，已经恢复了母突变株的生长缺陷是一个非常有用的方法，允许阐明重要和往往新颖的细胞过程10，11。
我们有兴趣控制谷氨酸平衡在B.亚提利斯12。与大肠杆菌类似，B. 亚提利通过抑制突变体的积累对谷氨酸平衡（即谷氨酸降解2中的块）的扰动做出反应。这些自发突变获得的抑制突变体的基因组变化被证明可以迅速恢复谷氨酸平衡9，13。因此，在细菌驯化过程中，B.亚提利适应特定生长状况，反映在酶合成和进化的酶活性中，这不足为奇，这些活动涉及谷氨酸代谢12。有人提出，在驯化过程中生长介质中缺乏外源性谷氨酸，是实验室菌株1682，14中神秘谷氨酸脱氢酶（GDH）gudB CR基因出现和固定的驱动力。这一假设得到我们观察的支持，即实验室菌株中GDH活性的减少为细菌提供了选择性生长优势，而外源性谷氨酸是稀缺的2。此外，在没有外源性谷氨酸的情况下，培育B.亚提利菌株，合成GDH GudB，导致抑制突变体的积累，这些突变体已经灭活了gadB基因2。显然，阴性活性GDH的存在对细胞不利，因为内源性产生的谷氨酸，否则可用于合成，会降解为铵和2-牛油酸盐（图1）。相比之下，当谷氨酸由介质提供时，配备高级 GDH 活性的B. 亚提利菌株比只合成一种功能性 GDH 的菌株具有选择性生长优势。有理由假设，高GDH活性允许细菌利用谷氨酸作为第二碳源，除了介质2提供的其他碳源（见图1）。因此，GDH 活性强烈地影响细菌的健身，具体取决于外源性谷氨酸的供应情况。
在这里，我们提出了一个非常说明性的方法来监测和可视化两个 B.亚蒂利斯 菌株之间的物种间竞争，在染色体上的单个轨迹不同（图2）。这两种菌株被标记为编码氟磷YFP和CFP的 yfp 和cfp的yfp和 cfp 基因，并在不同的营养条件下共同培养。通过随着时间的推移采样，并通过在阿加板块上电镀适当的稀释，每个培养物中的幸存者可以使用常见的立体荧光显微镜轻松监测。本文描述的程序是容易执行和适合可视化快速克隆扩张和消除有益和有害的突变，分别在细胞群随着时间的推移。

<table border="0" cellpadding="0" cellspacing="0" width="814">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">(NH4)2SO4&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3746</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Agar</td>
			<td style="width:175px;">Difco, USA</td>
			<td style="width:111px;">214010</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Ammonium ferric citrate (CAF)</td>
			<td rowspan="2" style="width:175px;">Sigma-Aldrich, Germany</td>
			<td rowspan="2" style="width:111px;">9714</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">CaCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">5239</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glucose</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3617</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Glycerol</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">4043</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">K2HPO4 x 3 H2O</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6878</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KCl</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3582</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">KH2PO4</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">3904</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">KOH</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">6751</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MgSO4 x 7 H2O&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">P027</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">MnCl2&#160;</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">T881</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">MnSO4 x 4 H2O</td>
			<td rowspan="2" style="width:175px;">Merck Millipore, Germany</td>
			<td rowspan="2" style="width:111px;">102786</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">NaCl</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">9265</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Nutrient broth</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">X929</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Potassium glutamate</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3712</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptone</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">8952</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Tryptophan</td>
			<td style="width:175px;">Applichem, Germany</td>
			<td style="width:111px;">A3445</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Yeast extract</td>
			<td style="width:175px;">Roth, Germany</td>
			<td style="width:111px;">2363</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,690,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">2.0 ml Reaction tubes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">72,691</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">15 ml Plastic tubes with screw cap</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">62,554,001</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Petri dishes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">82.1473</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">1.5 ml Polystyrene cuvettes</td>
			<td style="width:175px;">Sarstedt, Germany</td>
			<td style="width:111px;">67,742</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:363px;">15 ml Glass culture tubes&#160;</td>
			<td rowspan="2" style="width:175px;">Brand, Germany</td>
			<td rowspan="2" style="width:111px;">7790 22</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">with aluminium caps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">100 ml Shake flasks with aluminium caps</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">928 24</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Sterile 10 ml glass pipettes</td>
			<td style="width:175px;">Brand, Germany</td>
			<td style="width:111px;">278 23</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Incubator (28 and 37 &#176;C)</td>
			<td style="width:175px;">New Brunswick</td>
			<td style="width:111px;">M1282-0012</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="42">
			<td height="85" rowspan="3" style="height:85px;width:363px;">Standard pipette set (2-20 &#956;l, 10-100 &#956;l, 100-1000 &#956;l)</td>
			<td rowspan="3" style="width:175px;">Eppendorf, Germany</td>
			<td style="width:111px;">4910&#160;000.034, 4910 000.042,&#160;&#160;</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">4910 000.042,</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;">4910 000.069&#160;</td>
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Table top centrifuge for 1.5 and 2 ml reaction tubes</td>
			<td rowspan="2" style="width:175px;">Thermo Scientific, Heraeus Fresco 21, Germany</td>
			<td rowspan="2" style="width:111px;">75002425</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:363px;">Table top centrifuge for 15 ml plastic tubes</td>
			<td rowspan="2" style="width:175px;">Heraeus Biofuge Primo R, Germany</td>
			<td rowspan="2" style="width:111px;">75005440</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="80">
			<td height="101" rowspan="2" style="height:101px;width:363px;">Standard spectrophotometer</td>
			<td rowspan="2" style="width:175px;">Amersham Biosciences Ultrospec 2100 pro, Germany</td>
			<td rowspan="2" style="width:111px;">80-2112-21&#160;</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Stereofluorescence microscope&#160;</td>
			<td style="width:175px;">Zeiss SteREO Lumar V12, Germany</td>
			<td style="width:111px;">495008-0009-000</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Freezer (-20 and -80 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Fridge (4 &#176;C)</td>
			<td style="width:175px;">-</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Autoclave</td>
			<td style="width:175px;">Zirbus, LTA 2x3x4, Germany</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">pH meter</td>
			<td style="width:175px;">pH-meter 766, Calimatic, Knick, Germany</td>
			<td style="width:111px;">766</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="40">
			<td height="61" rowspan="2" style="height:61px;width:363px;">Vortex</td>
			<td rowspan="2" style="width:175px;">Vortex&#160; 3, IKA, Germany</td>
			<td rowspan="2" style="width:111px;">3340000</td>
			<td rowspan="2" style="width:167px;">-</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="21">
			<td height="64" rowspan="3" style="height:64px;width:363px;">Balance</td>
			<td rowspan="3" style="width:175px;">CP2202S, Sartorius, Germany</td>
			<td style="width:111px;">replaced by</td>
			<td rowspan="3" style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:111px;">CPA2202S</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:111px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Black pen (permanent marker)</td>
			<td style="width:175px;">Staedler, Germany</td>
			<td style="width:111px;">317-9</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Powerpoint program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Office Excel program</td>
			<td style="width:175px;">Microsoft, USA</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">Program for data processing</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:363px;">Adobe Photoshop CS5</td>
			<td style="width:175px;">Adobe, USA</td>
			<td style="width:111px;">replaced by CS6, download</td>
			<td style="width:167px;">Computer program for image processing</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:363px;">Computer</td>
			<td style="width:175px;">PC or Mac</td>
			<td style="width:111px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">ZEN pro 2011 software for the stereofluorescence microscope</td>
			<td style="width:175px;">Zeiss, Germany</td>
			<td style="width:111px;">410135 1002 110</td>
			<td style="width:167px;">AxioCam MRc Rev. Obtained through Zeiss</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Specific solution recipes</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">SP medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">8 g Nutrient broth</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.25 mg MgSO4 x 7 H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">1 g KCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid SP medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml CaCl2 (0.5 M), sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">1 ml MnCl2 (10 mM) sterilized by filtration</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">LB medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g Tryptone</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 g Yeast extract</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 g NaCl</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">if required, add 15 g agar for solid LB medium</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">C-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">CE-Glc minimal medium</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 ml 5 x C salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">10 ml III&#8217; salts</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">20 ml Glutamate (40%)</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">25 ml Glucose (20%), autoclaved for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">5 x C salts&#160;</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">20 g KH2PO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">80 g K2HPO4 x 3 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">16.5 g (NH4)2SO4</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">III&#8217; salts</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">0.232 g MnSO4 x 4 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">12.3 g MgSO4 x 7 H2O</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">40% Glutamate solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">200 g L-Glutamic acid</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">adjust the pH to 7.0 by adding approximately 80 g KOH</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">0.9% Saline (NaCl) solution</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 1 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">50% Glycerol solution</td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;width:815px;">295 ml Glycerol (87%)</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:23px;width:815px;">ad 0.5 l with sterile H2O, autoclave for 20 min at 121 &#176;C</td>
		</tr>
		<tr height="22">
			<td colspan="4" height="22" style="height:22px;width:815px;">Bacteria (All strains are based on the Bacillus subtilis strain 168)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP40 (rocG+ gudBCR amyE::PgudB-yfp)&#160;</td>
			<td rowspan="4" style="width:167px;">Laboratory strain collection</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP41 (rocG+ gudBCR amyE::PgudB-cfp)&#160;</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP52 (rocG+ gudB+ amyE::PgudB-cfp)</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;width:648px;">Bacillus subtilis BP156 (rocG+ gudB+ amyE::PgudB-yfp)</td>
		</tr>
	</tbody>
</table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

许多微生物，如细菌的增殖速度极快，种群可能达到高细胞密度。种群中很小一部分细胞总是积累的突变，这些突变对细胞有害或有益。如果突变的健身效应为亚人口提供了强大的选择性生长优势，这种亚种群的个体可能会迅速竞争，甚至完全消除其直接研究员。因此，小的基因变化和获得有益突变的细胞的选择驱动积累可能导致细胞种群的基因型完全改变。在这里，我们提出了一个程序，监测快速克隆扩张和消除有益和有害的突变，分别在细菌细胞群随着时间的推移，通过共生荧光标记个体的Gram阳性模型杆菌 亚蒂利斯。该方法易于执行，非常能说明细菌细胞群中个体之间的物种间竞争。

此处描述的方法成功地应用于可视化由 B. 亚蒂利斯 菌株组成的细胞群中的物种内部竞争，这些菌株分别标有编码荧光CFP和YFP的 cfp 和 yfp 基因。如 图3所示，该方法可用于以非常说明性的方式可视化物种内部竞争。通过在小区域发现样本，细胞群的克隆组成一目了然。虽然不适合定量分析，但这种方法有助于粗略估计不同生长参数（即氮源）对细胞群发育的...

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

细菌在有生之年可能会积累有害或有益的突变。在积累有益突变的细胞群中，个体可能迅速比其研究员竞争。在这里，我们介绍了一个简单的程序，以可视化物种内部竞争的细菌细胞种群随着时间的推移使用荧光标记的个人。

通过共生荧光标记菌株监测细菌细胞群中的物种内部竞争

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Research

JoVE Journal

Biology

8.4K 次观看.  Georg-August University. 细菌在有生之年可能会积累有害或有益的突变。在积累有益突变的细胞群中，个体可能迅速比其研究员竞争。在这里，我们介绍了一个简单的程序，以可视化物种内部竞争的细菌细胞种群随着时间的推移使用荧光标记的个人。

视频: Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Title Cell Encapsulation by Droplets

通过飞沫标题细胞封装

科研

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

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 ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3.
Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells.
Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11.
FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

A technique to probe the lipid raft partitioning of fluorescent proteins at the plasma membrane of living cells is described. It takes advantage of the disparity in diffusion times of proteins located inside or outside of lipid rafts. Acquisition can be performed dynamically in control conditions or after drug addition.

脂筏的测定分区在活细胞中的荧光标记探针的荧光相关光谱（FCS）

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析：在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

利用数字图像相关表征的血管组织标本本地菌株

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...